U.S. patent application number 16/885883 was filed with the patent office on 2020-12-24 for acoustic perfusion devices.
The applicant listed for this patent is FloDesign Sonics, Inc.. Invention is credited to Rudolf Gilmanshin, Thomas J. Kennedy, III, Bart Lipkens, Louis Masi, Erik Miller, Walter M. Presz, JR., Benjamin Ross-Johnsrud.
Application Number | 20200399583 16/885883 |
Document ID | / |
Family ID | 1000005062392 |
Filed Date | 2020-12-24 |
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United States Patent
Application |
20200399583 |
Kind Code |
A1 |
Lipkens; Bart ; et
al. |
December 24, 2020 |
ACOUSTIC PERFUSION DEVICES
Abstract
Methods are disclosed for separating beads and cells from a host
fluid. The method includes flowing a mixture containing the host
fluid, the beads, and the cells through an acoustophoretic device
having an ultrasonic transducer including a piezoelectric material
driven by a drive signal to create a multi-dimensional acoustic
standing wave. A drive signal is sent to drive the at least one
ultrasonic transducer to create the multi-dimensional acoustic
standing wave. A recirculating fluid stream having a tangential
flow path is located substantially tangential to the standing wave
and separated therefrom by an interface region. A portion of the
cells pass through the standing wave, and the beads are held back
from the standing wave in the recirculating fluid stream at the
interface region. Also disclosed is an acoustophoretic device
having a coolant inlet adapted to permit the ingress of a cooling
fluid into the device for cooling the transducer.
Inventors: |
Lipkens; Bart; (Bloomfield,
CT) ; Gilmanshin; Rudolf; (Framingham, MA) ;
Masi; Louis; (Del Mar, CA) ; Ross-Johnsrud;
Benjamin; (Northampton, MA) ; Miller; Erik;
(Belchertown, MA) ; Presz, JR.; Walter M.;
(Wilbraham, MA) ; Kennedy, III; Thomas J.;
(Wilbraham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FloDesign Sonics, Inc. |
Wilbraham |
MA |
US |
|
|
Family ID: |
1000005062392 |
Appl. No.: |
16/885883 |
Filed: |
May 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15689960 |
Aug 29, 2017 |
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16885883 |
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15420073 |
Jan 30, 2017 |
9745548 |
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15689960 |
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15139187 |
Apr 26, 2016 |
9752113 |
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15420073 |
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14975307 |
Dec 18, 2015 |
9822333 |
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15139187 |
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14175766 |
Feb 7, 2014 |
9416344 |
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14975307 |
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14026413 |
Sep 13, 2013 |
9458450 |
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14175766 |
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13844754 |
Mar 15, 2013 |
10040011 |
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14026413 |
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62307934 |
Mar 14, 2016 |
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62296685 |
Feb 18, 2016 |
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62288500 |
Jan 29, 2016 |
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62256952 |
Nov 18, 2015 |
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62243211 |
Oct 19, 2015 |
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62211057 |
Aug 28, 2015 |
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62093491 |
Dec 18, 2014 |
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61761717 |
Feb 7, 2013 |
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61708641 |
Oct 2, 2012 |
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61754792 |
Jan 21, 2013 |
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61708641 |
Oct 2, 2012 |
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61611240 |
Mar 15, 2012 |
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61611159 |
Mar 15, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/10 20130101;
C07K 1/14 20130101; B01D 17/04 20130101; C12N 13/00 20130101; C12M
29/18 20130101; B01D 21/283 20130101; G10K 15/043 20130101; B06B
1/0644 20130101; B01D 17/06 20130101; C12M 35/04 20130101; C12M
41/12 20130101; C12M 33/08 20130101; C12M 29/10 20130101; G10K
15/00 20130101; C12M 47/02 20130101; G10K 9/122 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; B01D 21/28 20060101 B01D021/28; C12M 1/34 20060101
C12M001/34; G10K 15/04 20060101 G10K015/04; G10K 9/122 20060101
G10K009/122; C12M 1/26 20060101 C12M001/26; C12N 13/00 20060101
C12N013/00; C07K 1/14 20060101 C07K001/14; C12M 1/42 20060101
C12M001/42; B01D 17/06 20060101 B01D017/06; B06B 1/06 20060101
B06B001/06; B01D 17/04 20060101 B01D017/04; G10K 15/00 20060101
G10K015/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
No. IIP-1330287 (Amendment 003, Proposal No. 1458190) awarded by
the National Science Foundation. The government has certain rights
in the invention.
Claims
1. An acoustic bead retention device, comprising: a chamber; at
least one ultrasonic transducer coupled to the chamber; the at
least one ultrasonic transducer configured to be excited to
generate a multi-dimensional acoustic standing wave that is
configured to retain beads and pass fluid in which the beads are
entrained.
2. The device of claim 1, further comprising a recirculation path
communicating with the acoustic chamber.
3. The device of claim 1, wherein the multi-dimensional acoustic
standing wave is further configured to generate a pressure rise and
an acoustic radiation force on the beads at an interface region of
the multi-dimensional acoustic standing wave.
4. The device of claim 3, further comprising a locale adjacent to
the interface region where the bead concentration is increased.
5. The device of claim 1, further comprising a locale in the
multi-dimensional acoustic standing wave where the bead
concentration is increased.
6. The device of claim 1, wherein the multi-dimensional acoustic
standing wave is further configured to generate an acoustic
radiation force with an axial force component and a lateral force
component that are of the same order of magnitude.
7. The device of claim 1, further comprising a control system
coupled to the at least one ultrasonic transducer to obtain
feedback for control of the at least one ultrasonic transducer.
8. The device of claim 7, further comprising a flow rate monitor
coupled to the control system to obtain feedback for control of the
flow rate.
9. The device of claim 1, further comprising an inlet communicating
with the acoustic chamber for providing media to the chamber to
permit media exchange for or washing of the retained beads.
10. The device of claim 9, further comprising an outlet
communicating with the chamber for withdrawing the retained
beads.
11. A method for separating biological material in a fluid,
comprising: providing beads that are functionalized with a material
with an affinity for the biological material to the fluid; flowing
the fluid containing the biological material through an acoustic
retention device, the device comprising: a chamber; at least one
ultrasonic transducer coupled to the chamber; the at least one
ultrasonic transducer configured to be excited to generate a
multi-dimensional acoustic standing wave; and exciting the at least
one ultrasonic transducer to generate the multi-dimensional
acoustic standing wave; and retaining the beads with the
multi-dimensional acoustic standing wave; and capturing the
biological material with the beads.
12. The method of claim 11, further comprising recirculating the
fluid containing the biological material to the chamber.
13. The method of claim 11, further comprising generating a
pressure rise and an acoustic radiation force on the beads at an
interface region of the multi-dimensional acoustic standing
wave.
14. The method of claim 13, further comprising increasing bead
concentration adjacent to the interface region.
15. The method of claim 11, further comprising increasing bead
concentration in the multi-dimensional acoustic standing wave.
16. The method of claim 11, further comprising exciting the at
least one ultrasonic transducer to generate the multi-dimensional
acoustic standing wave with an axial force component and a lateral
force component that are of the same order of magnitude.
17. The method of claim 11, further comprising: obtaining feedback
from the at least one ultrasonic transducer; and controlling the at
least one ultrasonic transducer in accordance with the
feedback.
18. The method of claim 11, further comprising flowing media
through the chamber while the beads with the captured biological
material are retained to perform a media exchange for or washing of
the biological material.
19. A method for providing a media exchange for cells or for
washing cells, comprising: capturing the cells with beads that are
functionalized with an affinity for the cells; retaining the beads
with the captured cells using the acoustic bead retention device of
claim 1; and flowing a fluid into the chamber and through the
multi-dimensional acoustic standing wave.
20. A method for retaining material in a fluid, comprising:
generating a multi-dimensional acoustic standing wave with an
acoustic transducer; trapping and clustering the material in the
multi-dimensional acoustic standing wave to permit the material to
grow in size and exit the multi-dimensional acoustic standing wave;
and generating an acoustic edge effect with the multi-dimensional
acoustic standing wave to form a barrier to entry to the
multi-dimensional acoustic standing wave for the material.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/689,960, filed Aug. 29, 2017, which is a
continuation-in-part of U.S. patent application Ser. No.
15/420,073, filed Jan. 30, 2017, which claims priority to U.S.
Provisional Patent Application Ser. No. 62/288,500, filed Jan. 29,
2016, and to U.S. Provisional Patent Application Ser. No.
62/296,685, filed Feb. 18, 2016, and to U.S. Provisional Patent
Application Ser. No. 62/307,934, filed Mar. 14, 2016. U.S. patent
application Ser. No. 15/420,073 is also a continuation-in-part of
U.S. patent application Ser. No. 15/139,187, filed Apr. 26, 2016,
which is a continuation-in-part of U.S. patent application Ser. No.
14/975,307, filed Dec. 18, 2015, which claims priority to U.S.
Provisional Patent Application Ser. No. 62/256,952, filed on Nov.
18, 2015, and to U.S. Provisional Patent Application Ser. No.
62/243,211, filed on Oct. 19, 2015, and to U.S. Provisional Patent
Application Ser. No. 62/211,057, filed on Aug. 28, 2015, and to
U.S. Provisional Patent Application Ser. No. 62/093,491, filed on
Dec. 18, 2014. U.S. patent application Ser. No. 14/975,307 is also
a continuation-in-part of U.S. patent application Ser. No.
14/175,766, filed on Feb. 7, 2014, which claims priority to U.S.
Provisional Patent Application Ser. No. 61/761,717, filed on Feb.
7, 2013, and is also a continuation-in-part of U.S. patent
application Ser. No. 14/026,413, filed on Sep. 13, 2013, which
claims the benefit of U.S. Provisional Patent Application Ser. No.
61/708,641, filed on Oct. 2, 2012. U.S. patent application Ser. No.
14/026,413 is also a continuation-in-part of U.S. Ser. No.
13/844,754, filed Mar. 15, 2013, which claims the benefit of U.S.
Provisional Patent Application Ser. No. 61/611,159, filed Mar. 15,
2012, and of U.S. Provisional Patent Application Ser. No.
61/611,440, filed Mar. 15, 2012, and of U.S. Provisional Patent
Application Ser. No. 61/754,792, filed Jan. 21, 2013, and of U.S.
Provisional Patent Application Ser. No. 61/708,641, filed on Oct.
2, 2012. These applications are hereby incorporated herein by
reference in their entireties.
BACKGROUND
[0003] The field of biotechnology has grown tremendously in the
last 20 years. This growth has been due to many factors, some of
which include the improvements in the equipment available for
bioreactors, the increased understanding of biological systems and
increased knowledge as to the interactions of materials (such as
monoclonal antibodies and recombinant proteins) with the various
systems of the human body.
[0004] A perfusion bioreactor processes a continuous supply of
fresh media that is fed into the bioreactor while growth-inhibiting
byproducts are constantly removed. The nonproductive downtime can
be reduced or eliminated with a perfusion bioreactor process. The
cell densities achieved in perfusion culture (30-100 million
cells/mL) are typically higher than for fed-batch modes (5-25
million cells/mL). These improvements have led to lower
contamination in the harvest and better yields without significant
increase in cost. A perfusion bioreactor may use a cell retention
device to prevent escape of the culture when byproducts are being
removed. These cell retention systems may add a level of complexity
to the perfusion process, where further management, control, and/or
maintenance operations may be implemented. Operational issues such
as malfunction or failure of the cell retention equipment has
previously been a problem with perfusion bioreactors, which has
limited their attractiveness in the past.
[0005] In the biotechnology and biopharma fields, it is desirable
to separate many different types of materials from a primary fluid
stream based on size, surface active materials, density, and other
characteristics. Tangential flow filtration has been widely adopted
to separate and concentrate diverse array of microbes and other
materials from a fluid stream. These materials have been filtered
utilizing different physical polymer membranes such as
polyvinylidene difluoride (PDF) and polyether sulfone (PES).
[0006] Issues that arise with tangential flow filtration include
the cost of the membrane materials, the formation of a gel layer or
filtrate-suppressing layer on the filter membranes, the consistency
of the polymer membranes, and the entire efficiency of the process.
There is also the possibility of product loss due to clogging of
the polymer membranes and the need to replace these clogged
membranes.
[0007] There is therefore a need to improve the continuous
processing of materials throughout the tangential flow process,
decrease the cost, increase the efficiency, and improve the overall
ability of the separation process to perform primary, secondary,
tertiary and beyond separation of materials from a primary fluid
stream.
BRIEF DESCRIPTION
[0008] The present disclosure relates, in various embodiments, to
acoustic devices which are used for perfusion biomanufacturing.
More particularly, the devices are coupled to an associated
bioreactor. Within the bioreactor, biomolecules, such as
recombinant proteins or monoclonal antibodies, and other materials
are produced. Materials may include viruses, virus-like particles,
exosomes, oncozomes, materials from cell free protein synthesis,
cell vesicles, proteins, monoclonal antibodies, recombinant
proteins and other materials. The acoustic device is used for
separating desirable products from the bioreactor contents on a
continuous basis, and the cells or other materials are continuously
returned to the bioreactor for further culturing. Generally, a
fluid mixture containing the cells and the desired products are
passed or flowed through the acoustic device and separated therein
by multi-dimensional standing wave(s). The fluid mixture generally
also contains other materials, such as cell debris and fines. The
fluid mixture can be continuously flowed into the device, with
desired products being continuously removed. The acoustic perfusion
device returns healthy viable cells to the bioreactor while desired
products are harvested and flowed downstream for further
processing, e.g., additional filtering, chromatography, etc.
Additionally, the cell culture media in the bioreactor is clarified
as cell fragments are also allowed to pass into the harvest stream
and thereby out of the fluid mixture being recycled to the
bioreactor. This arrangement results in lower overall cell culture
media usage, corresponding to a predicted cost savings of up to
$20,000 per day for large bioreactors.
[0009] Disclosed in various embodiments are acoustic perfusion
devices, comprising: an acoustic chamber; an inlet port, an inlet
flow path leading from the inlet port to the acoustic chamber; an
outlet port for recirculating fluid flowing through the device back
to its source (e.g. a bioreactor), and an outlet flow path leading
from the acoustic chamber to the outlet port; at least one
collection or harvest port for collecting a product stream of fluid
exiting the acoustic chamber; and at least one ultrasonic
transducer in the acoustic chamber below the at least one harvest
port, the at least one ultrasonic transducer including a
piezoelectric material driven by a drive signal to create an
acoustic standing wave across a collection or harvest flow path
leading from the acoustic chamber to the at least one collection or
harvest port. The acoustic standing wave may be planar or
multi-dimensional, or a combination of such waves may be present
within the acoustic chamber (generally from multiple transducers).
The acoustic standing wave can be thought of as a "force field"
that holds back whole cells but permits smaller materials such as
the desired biomolecules (e.g. recombinant proteins and/or
monoclonal antibodies) and cell fragments, to pass through and be
removed from the fluid that is returned to the bioreactor.
[0010] The outlet port is generally below the inlet port, and is
generally located at a bottom end of the device.
[0011] As mentioned above, the device may have one or more
collection or harvest ports at the top of the device. In some more
specific embodiments, the device may have a total of two harvest
ports spaced apart from each other on the top end of the
device.
[0012] In particular embodiments, the inlet port is at a first end
of the device at a first height, the at least one ultrasonic
transducer is at a second height above the first height, and a
bottom wall extends from the inlet port to the outlet port. The
outlet port may be located at a second end of the device opposite
the first end. The bottom wall may be concave, relative to a line
between the inlet port and the outlet port. The device may include
an upper wall above the inlet flow path. The inlet port, the outlet
port, and the at least one harvest port are sometimes all located
on a front wall of the device. The front wall itself may be planar
(i.e. flat).
[0013] The device can further comprise a reflector located in the
acoustic chamber opposite the at least one ultrasonic transducer.
Alternatively, the device can have a total of two ultrasonic
transducers located on opposite sides of the harvest flow path at
the same height and facing each other, or additional ultrasonic
transducers can be located on multiple sides of the
collection/harvest flow path. A reflector may be located between
the two ultrasonic transducers. There may also be a plurality of
transducer/reflector pairs located as appropriate to form planar,
multi-dimensional, or combinations of such acoustic standing
wave(s).
[0014] In particular embodiments, the acoustic standing wave
results in an acoustic radiation force having an axial force
component and a lateral force component that are of the same order
of magnitude.
[0015] In other embodiments of the device disclosed herein, the
inlet flow path leads from the inlet port downwards towards a
bottom end of the device and past the outlet port, and then upwards
to the acoustic chamber. Sometimes, the inlet port and the at least
one harvest port are both located on a top wall of the device, and
the outlet port is located on a front wall of the device. The at
least one ultrasonic transducer may be mounted in a rear wall or a
front wall of the device. The bottom wall of this acoustic chamber
can be a sloped planar surface. The reflector may be made of a
transparent material.
[0016] The inlet flow path may be shaped to generate a tangential
flow path below an acoustic field generated by the acoustic
standing wave. In still additional versions seen herein, the inlet
flow path enters the acoustic chamber on a first side of the
device, and the outlet port is located (i) on the first side of the
device or (ii) on a second opposite side. The inlet port can be
located on a front side of the device, and the at least one harvest
port can be located on a top wall of the device. The at least one
transducer can be located on a front side or a rear side of the
device. In more particular embodiments, there can be two
transducers, one on the front side and one of the rear side. In yet
other particular embodiments, there is an ultrasonic transducer on
the front or rear side, and a reflector located on the respective
rear or front side opposite the transducer.
[0017] In additional embodiments, the perfusion device further
comprises a recirculation flow path between the inlet port and the
outlet port that does not enter the acoustic chamber, and the
recirculation flow path is located below the acoustic chamber. In
some particular embodiments, the inlet flow path travels through a
different passage than the outlet flow path. In yet other
embodiments, the inlet flow path and the outlet flow path travel
through a common passage.
[0018] The device may be attached to a mounting piece having holes
for attachment.
[0019] Also disclosed are methods for separating cells from a fluid
mixture containing the cells. The fluid mixture is flowed through
an acoustic perfusion device of the structure described above,
having at least one ultrasonic transducer. The at least one
ultrasonic transducer is driven to create the acoustic standing
wave. A fluid enriched in cells can be collected from the outlet
port and a clarified fluid, depleted in cells, can be collected
from the at least one harvest port.
[0020] In particular embodiments, the flow rate through the
collection/harvest flow path is at least one order of magnitude
smaller than a flow rate through the inlet flow path. In specific
embodiments, a flow rate of the fluid mixture entering the device
through the inlet port is about 1 liter per minute and a flow rate
of the fluid depleted in cells exiting the device through the at
least one collection/harvest port is about 10 milliliters per
minute. Alternatively, the ratio of the flow rate entering through
the inlet port to the flow rate exiting through the at least one
collection/harvest port is such that the acoustic standing wave is
not overcome by the main body of cells, or in other words so that a
large volume of cells do not begin exiting the device through the
collection/harvest port(s).
[0021] The methods may further comprise pulling the fluid mixture
through the device using a first pump attached to the at least one
harvest port of the device and a second pump attached to the outlet
port of the device.
[0022] Also disclosed herein are flow devices adapted to (i)
receive a flowing mixture containing a primary fluid and cells; and
(ii) to use a first acoustic standing wave to continuously draw off
a harvest fluid stream depleted in cells from the flowing mixture,
thereby changing the cell concentration of the flowing mixture. A
pressure rise may be generated on the upstream interface region of
the acoustic standing wave, along with an acoustic radiation force
acting on the incoming suspended particles. This "interface
effect", which may also be termed "edge effect", acts as a barrier
and is typically located at the upstream bounding surface of the
volume of fluid that is ensonified by the transducer (i.e. the flow
mixture crosses the interface region to enter the ensonified volume
of fluid). The frequency of the acoustic standing wave may be
modified such that different contrast factor materials may be held
back or allowed through the acoustic standing wave, or such that
particles of one given size range are retained and particles of a
second given range are allowed to flow through the standing
wave.
[0023] The device may further comprise a secondary flow chamber in
which the harvest fluid stream depleted in cells passes through a
second acoustic standing wave having a frequency different from, or
equal to the first acoustic standing wave. For example, the second
acoustic standing wave may have a higher or lower frequency than
the first acoustic standing wave. The ratio of the frequency of the
two standing waves is, in some embodiments, at least 2:1 (i.e. one
of the frequencies is at least twice the other frequency, e.g. 3
MHz and 6 MHz).
[0024] Also disclosed herein are flow devices that comprise: at
least one inlet for receiving a flowing mixture of a primary fluid
and cells, an ultrasonic transducer that produces a first
ultrasonic acoustic standing wave and uses a pressure rise and an
acoustic radiation force generated on an upstream interface region
of the first ultrasonic acoustic standing wave to separate the
flowing mixture into a primary high cell concentration fluid stream
and a secondary harvest fluid stream; an outlet port for the
primary high cell concentration fluid stream; and at least one
collection port for the secondary harvest fluid stream. A bleed
port can also be present for extracting a concentrated fluid/cell
mixture. The fluid mixture may comprise particles such as mammalian
cells, bacteria, cell debris, fines, proteins, exosomes, vesicles,
viruses, and insect cells.
[0025] The device may further comprise a secondary flow chamber in
which the secondary harvest fluid stream passes through a second
acoustic standing wave having a frequency different from, or equal
to, the first ultrasonic acoustic standing wave.
[0026] Also disclosed herein are methods for separating
microspheres, microbeads, nano beads, microcarriers and other micro
and nano particulates (collectively known as beads), and cells from
a host fluid. The methods comprise flowing a mixture containing a
host fluid, the beads, and the cells through an acoustophoretic
device; sending a voltage signal to drive an ultrasonic transducer
to create the multi-dimensional acoustic standing wave, wherein a
recirculating fluid stream having a tangential flow path is located
substantially tangential to the multi-dimensional acoustic standing
wave and separated therefrom by an interface region; and wherein at
least a portion of the cells (e.g., at least 95% of the cells,
including up to about 99% of the cells) pass through the
multi-dimensional acoustic standing wave, and the beads are held
back from the multi-dimensional acoustic standing wave in the
recirculating fluid stream at the interface region. The
acoustophoretic device may be constructed as described herein,
namely comprising a flow chamber having at least one inlet and at
least one outlet; at least one ultrasonic transducer located in the
flow chamber, the at least one ultrasonic transducer including a
piezoelectric material driven by a voltage signal to create a
multi-dimensional acoustic standing wave in the flow chamber; and a
reflector located in the flow chamber opposite from the at least
one ultrasonic transducer.
[0027] In particular embodiments, the beads are non-functionalized.
Other embodiments include functionalizing the beads such that the
beads attach to the cells. The beads can be functionalized with
various materials, such as antigens, on the surface that will allow
for affinity binding of materials (e.g., viruses, virus-like
particles, exosomes, oncozomes, materials from cell free will
protein synthesis, proteins, monoclonal antibodies, recombinant
proteins) to the beads. The functionalized beads may, in some
embodiments, have a positive contrast factor. The functionalized
beads having a positive contrast factor may be selected from the
group consisting of polystyrene beads and glass beads. In other
embodiments, have a negative contrast factor. The functionalized
beads having a negative contrast factor may be selected from the
group consisting of microbubbles and micro-glass spheres. The beads
may, in certain embodiments, be polymeric, glass, hollow, or
gas-filled. The beads can be spherical, toroidal, cylindrical, or
conical.
[0028] In certain embodiments of the method, the mixture can
include microvesicles (e.g., exosomes, oncosomes, viruses,
proteins, recombinant proteins, and monoclonal antibodies). A
pressure rise and an acoustic radiation force on cells can be
generated at the interface region to clarify the host fluid as it
passes through the multi-dimensional acoustic standing wave. In
particular embodiments, the method further comprises collecting the
cells after passing through the multi-dimensional acoustic standing
wave for a first time and recirculating the collected cells are
back through the device for separation by the multi-dimensional
acoustic standing wave for a second time. The method can further
comprise collecting the cells after passing through the
multi-dimensional acoustic standing wave for the second time.
[0029] Also disclosed herein are acoustophoretic devices including
a flow chamber having at least one inlet port and at least one
outlet; at least one ultrasonic transducer coupled to the flow
chamber and at least one reflector coupled to the flow chamber
opposite the at least one ultrasonic transducer, wherein the at
least one ultrasonic transducer includes a piezoelectric material
driven by a voltage signal to create a multi-dimensional acoustic
standing wave in the device; and a coolant inlet adapted to permit
the ingress of an associated cooling fluid into the device for
cooling the at least one ultrasonic transducer.
[0030] In certain constructions, the at least one inlet port
includes a first inlet port on a front wall of the device, a second
inlet port on a rear wall of the device opposite the front wall
thereof, and the at least one outlet includes a permeate outlet on
the first sidewall of the device, a wastewater outlet on the first
sidewall of the device, and a drain outlet on a first sidewall of
the device.
[0031] The cooling fluid can be the same or different from the
primary fluid that flows through the flow chamber carrying
particles (e.g., cells and beads). For example, the cooling fluid
can be water, air, alcohol, ethanol, ammonia, or some combination
thereof. The cooling fluid can, in certain embodiments, be a
liquid, gas, or gel. The cooling fluid can be an electrically
non-conductive fluid to prevent electric short-circuits.
[0032] These and other non-limiting characteristics are more
particularly described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The following is a brief description of the drawings, which
are presented for the purposes of illustrating the exemplary
embodiments disclosed herein and not for the purposes of limiting
the same.
[0034] FIG. 1 illustrates a single standing acoustic wave generated
by an ultrasonic transducer and a reflector.
[0035] FIG. 2 is an illustration comparing a fed-batch bioreactor
system with a perfusion bioreactor system.
[0036] FIG. 3 is a cross-sectional view that shows the various
components of a stirred-tank bioreactor.
[0037] FIG. 4 is a perspective view of one exemplary embodiment of
an acoustic perfusion device of the present disclosure, having two
collection or harvest ports and a single ultrasonic transducer.
[0038] FIG. 5 shows a second exemplary embodiment of an acoustic
perfusion device of the present disclosure, with a single reflector
located between two ultrasonic transducers.
[0039] FIG. 6 is a schematic view illustrating a perfusion
bioreactor coupled with an acoustic perfusion device of the present
disclosure, and a recycle path.
[0040] FIG. 7 is a front cross-sectional view of a third exemplary
embodiment of an acoustic perfusion device of the present
disclosure.
[0041] FIG. 8 is an exterior perspective view of the acoustic
perfusion device of FIG. 7.
[0042] FIG. 9 is a front cross-sectional view of a fourth exemplary
embodiment of an acoustic perfusion device of the present
disclosure.
[0043] FIG. 10 is a perspective view of the acoustic perfusion
device of FIG. 9.
[0044] FIG. 11 is a cross-sectional diagram of a conventional
ultrasonic transducer.
[0045] FIG. 12 is a cross-sectional diagram of an ultrasonic
transducer of the present disclosure. An air gap is present within
the transducer, and no backing layer or wear plate are present.
[0046] FIG. 13 is a cross-sectional diagram of an ultrasonic
transducer of the present disclosure. An air gap is present within
the transducer, and a backing layer and wear plate are present.
[0047] FIG. 14 is a graph of electrical impedance amplitude versus
frequency for a square transducer driven at different
frequencies.
[0048] FIG. 15 illustrates the trapping line configurations for
seven of the resonance frequencies (minima of electrical impedance
amplitudes) of FIG. 14 from the direction orthogonal to fluid
flow.
[0049] FIG. 16 is a computer simulation of the acoustic pressure
amplitude (right-hand scale in Pa) and transducer out of plane
displacement (left-hand scale in meters). The text at the top of
the left-hand scale reads ".times.10.sup.-7". The text at the top
of the left-hand scale by the upward-pointing triangle reads
"1.473.times.10.sup.-6". The text at the bottom of the left-hand
scale by the downward-pointing triangle reads
"1.4612.times.10.sup.-10". The text at the top of the right-hand
scale reads ".times.10.sup.6". The text at the top of the
right-hand scale by the upward-pointing triangle reads
"1.1129.times.10.sup.6". The text at the bottom of the right-hand
scale by the downward-pointing triangle reads "7.357". The
triangles show the maximum and minimum values depicted in this
figure for the given scale. The horizontal axis is the location
within the chamber along the X-axis, in inches, and the vertical
axis is the location within the chamber along the Y-axis, in
inches.
[0050] FIG. 17 shows the In-Plane and Out-of-Plane displacement of
a crystal where composite waves are present.
[0051] FIG. 18 is a view of a first acoustic perfusion device of
the present disclosure fluidly connected to an associated
bioreactor, showing a plurality of hoses fluidly connecting the
various ports of the device to the associated bioreactor and an
outflow pump fluidly connecting the outlet port of the device to
the associated bioreactor.
[0052] FIG. 19 is a view of another acoustic perfusion device of
FIG. 5, showing a reflector in the acoustic chamber between first
and second transducers. A fluid mixture is also present in the
device and arrows are shown indicating the direction of flow in
addition to waves indicating the acoustic field between the
reflector and first and second transducers.
[0053] FIG. 20 is a graph showing the efficiency of removing cells
from a fluid mixture for one experiment at two different
perfusate/feed rates.
[0054] FIG. 21 is a graph showing the harvest flow (also referred
to as the perfusate) turbidity reduction for an experiment
[0055] FIG. 22 is a graph showing the cell viability for varied
flow rates for the experiment conducted for the graphs of FIGS.
20-21.
[0056] FIG. 23 is a graph showing the total cell density and cell
retention for varied flow rates and flow methods for another
experiment.
[0057] FIG. 24 is a graph showing the cell viability for varied
flow rates for the experiment conducted for the graphs of FIG.
23.
[0058] FIG. 25 is a graph showing the total cell density and cell
retention for varied numbers of ultrasonic transducers for another
experiment.
[0059] FIG. 26 is a graph showing the cell viability for varied
numbers of ultrasonic transducers for the experiment conducted for
the graph of FIG. 25.
[0060] FIG. 27 is a picture of another acoustic perfusion device
that was tested.
[0061] FIG. 28 is a graph showing the effect of the perfused flow
rate or the transducer voltage on the cell retention.
[0062] FIG. 29A is a microscope image from a ViCell Cell Analyzer
of the particles in the feed stream going into the device. The feed
stream is a bioreactor fluid containing CHO cells, protein, and
cell fragments. FIG. 29B is a graph of the particle diameter
distribution of the feed, showing a bimodal distribution. In FIG.
29B, the y-axis is the particle count from 0 to 200 in intervals of
10. The x-axis is the particle diameter in microns from 6 to 50 in
intervals of 2. The total number of particles is 5539, the mean
particle size is 16.78 microns, standard deviation of 6.76 microns,
and mode particle size of 10.56 microns.
[0063] FIG. 30A is a microscope image of the perfusate (or
clarified harvest flow) exiting the device. FIG. 30B is a graph of
the particle diameter distribution of the perfusate, showing a
unimodal distribution at much lower sizes. In FIG. 30B, the y-axis
is the particle count from 0 to 300 in intervals of 20. The x-axis
is the particle diameter in microns from 6 to 50 in intervals of 2.
The total number of particles is 2919, the mean particle size is
10.08 microns, standard deviation of 3.75 microns, and mode
particle size of 8.99 microns.
[0064] FIG. 31 is a CFD model showing the velocity distribution
within the device of FIG. 27. The text at the top of the scale
reads ".times.10.sup.-2". The text at the top of the scale by the
upward-pointing triangle reads "0.678". The text at the bottom of
the scale by the downward-pointing triangle reads "0". The
triangles show the maximum and minimum values depicted in this
figure for the given scale. The scale runs from 0 to 5 m/s in
intervals of 0.5, with black indicating 5 at the top of the scale,
and white indicating zero at the bottom of the scale.
[0065] FIG. 32 is a front view of the device of FIG. 27, showing
the flow paths, acoustic field, and acoustic interface effect.
[0066] FIG. 33 is a composite photograph showing the acoustic
perfusion device of FIG. 27 in two operating modes. On the left,
the device is in startup or cell settling mode. On the right, the
device is in steady cell retention mode.
[0067] FIG. 34 shows the geometry of a model simulation of the
acoustic device used for cell retention. The model contains two
fluids, one a clarified fluid within the acoustic field, the other
a high cell density fluid to the left of the acoustic field, a
piezoelectric transducer, a steel reflector, and an aluminum
housing. The first fluid was water within the acoustic field and
the second fluid was a 15% concentration of CHO cells in water
solution outside (to the left) of the acoustic field. The blue
solid line in the model indicates the separation line between the
two fluids.
[0068] FIGS. 35A, 35B, and 35C are graphs showing the displacement
of the piezoelectric material, the aluminum housing, and the steel
reflector (left-side scale); and the acoustic pressure in the two
fluids (right-side scale) of the model simulation of FIG. 34 at
several frequencies of operation. FIG. 35A is at a frequency of
2.218 MHz.
[0069] FIG. 35B is at a frequency of 2.2465 MHz. FIG. 35C is at a
frequency of 2.3055 MHz. For all three graphs, the left-side scale
is indicated with text at the top of the scale reading
".times.10.sup.-6" or ".times.10.sup.-7", and is in units of
inches. The right-side scale is indicated with text at the top of
the scale reading ".times.10.sup.6", and is in units of Pascals.
The y-axis runs from -0.8 to 1.6 in intervals of 0.2. The x-axis
runs from -0.5 to 1.5 in intervals of 0.5.
[0070] FIG. 36 is a graph showing the average lateral force (N) and
the average lateral force normalized by power (N/W) acting on
suspended CHO cells at several frequencies of operation.
[0071] FIG. 37 is a picture (top view) of an acoustic perfusion
device of the present disclosure. Arrows indicate the flow into the
inlet port; the flow out of the outlet port; the clarified fluid
flow out the top of the device and the flow of concentrate out the
bottom of the device.
[0072] FIG. 38 is a picture (side view) of the acoustic perfusion
device of FIG. 37.
[0073] FIG. 39 is a graph of cell retention vs. perfusate flowrate
for the device of FIG. 37.
[0074] FIG. 40 is a prior art illustration showing direct flow
filtration (DFF) and tangential flow filtration (TFF).
[0075] FIG. 41 is a picture illustrating a first mode of operation
during perfusion, in which cells are trapped, clustered, and
separated from a harvest stream. The device is operated vertically,
with an arrow indicating the direction of gravity.
[0076] FIG. 42 is a picture illustrating a second mode of operation
during perfusion, in which cells are prevented from entering an
acoustic standing wave field while smaller particles are permitted
to pass through the field and into the harvest stream. The device
is operated vertically, with an arrow indicating the direction of
gravity.
[0077] FIG. 43 is a perspective view of a fifth exemplary
embodiment of an acoustic perfusion device of the present
disclosure. This embodiment includes a direct recirculation flow
path between the inlet port and the outlet port. An inflow
passageway and an outflow passageway join the recirculation flow
path to the acoustic chamber, and create a tangential sweeping flow
underneath the acoustic field.
[0078] FIG. 44 is a front view picture of the device of FIG. 43.
The inflow passageway and the outflow passageway are clearly
visible, along with the recirculation pipe.
[0079] FIG. 45 is a diagrammatic side view of the device of FIG.
43.
[0080] FIG. 46A and FIG. 46B are a diagrammatic front view of the
device of FIG. 43, showing one internal structure. The figures are
duplicated due to the number of reference numerals.
[0081] FIG. 46C and FIG. 46D are a diagrammatic front view of the
device of FIG. 43, showing an alternative internal structure, where
the inflow passageway and the outflow passageway have a different
flow geometry. The figures are duplicated due to the number of
reference numerals.
[0082] FIG. 47 is a perspective view of a sixth exemplary
embodiment of an acoustic perfusion device of the present
disclosure. This embodiment includes a direct recirculation flow
path between the inlet port and the outlet port. A single
passageway joins the recirculation flow path to the acoustic
chamber, and acts as both the inflow passageway and the outflow
passageway.
[0083] FIG. 48 is a diagrammatic side view of the device of FIG.
43.
[0084] FIG. 49 is a diagrammatic front view of the device of FIG.
43, showing the internal structure.
[0085] FIG. 50 is a graph of cell retention versus time for the
device of FIG. 7. The y-axis runs from 90% to 100% in intervals of
1%. The x-axis runs from 0 to 165 minutes in intervals of 15
minutes. Tests were performed on two different days.
[0086] FIG. 51 is a graph of perfusate cell density (million
cells/mL) versus time for the device of FIG. 7. The y-axis runs
from 0.00 to 3.50 in intervals of 0.50. The x-axis runs from 0 to
165 minutes in intervals of 15 minutes. Tests were performed on two
different days.
[0087] FIG. 52 is a CFD model showing the velocity distribution
within the device of FIG. 46A. The text at the top of the scale
reads ".times.10.sup.-3". The scale runs from -5 to 5 m/s in
intervals of 1.
[0088] FIG. 53 is a CFD model showing the velocity distribution
within the device of FIG. 46C at 250 mL/min flowrate. The text at
the top of the scale reads ".times.10.sup.-3". The scale runs from
-5 to 5 m/s in intervals of 1.
[0089] FIG. 54 is a CFD model showing the velocity distribution
within the device of FIG. 46C at 1000 mL/min flowrate. The text at
the top of the scale reads ".times.10.sup.-3". The scale runs from
-5 to 5 m/s in intervals of 1.
[0090] FIG. 55 is a CFD model showing the velocity distribution for
another internal structure for the device of FIG. 43. The scale
runs from -0.05 to 0.1 m/s in intervals of 0.05.
[0091] FIG. 56 is a CFD model showing the velocity distribution for
another internal structure for the device of FIG. 43. The scale
runs from -0.1 to 0.15 m/s in intervals of 0.05.
[0092] FIG. 57 is a graph of cell retention versus time for the
device of FIG. 46A. The y-axis runs from 90% to 100% in intervals
of 1%. The x-axis runs from 0 to 100 minutes in intervals of 10
minutes.
[0093] FIG. 58 is a graph of perfusate cell density (million
cells/mL) versus time for the device of FIG. 46A. The y-axis runs
from 0.00 to 1.20 in intervals of 0.20. The x-axis runs from 0 to
100 minutes in intervals of 10 minutes.
[0094] FIG. 59 is a graph of cell retention versus time for the
device of FIG. 46A at two different frequencies, 1 MHz and 2 MHz.
The darker circles are for 2 MHz. The y-axis runs from 0.00 to 1.00
in intervals of 0.10. The x-axis runs from 0 to 300 minutes in
intervals of 50 minutes.
[0095] FIG. 60 is a graph of perfusate cell density (million
cells/mL) versus time for the device of FIG. 46A at two different
frequencies, 1 MHz and 2 MHz. The darker circles are for 2 MHz. The
y-axis runs from 0.00 to 25.00 in intervals of 5.00. The x-axis
runs from 0 to 300 minutes in intervals of 50 minutes.
[0096] FIG. 61 is a front cross-sectional view of a seventh
exemplary embodiment of an acoustic perfusion device of the present
disclosure.
[0097] FIG. 62 is a front view photograph of a working 1 inch by 1
inch by 1 inch separator system using a 1 inch by 1 inch crystal to
separate beads from a yeast mixture.
[0098] FIG. 63 is a log-log graph showing the relationship of the
MRC-5 radiation force, the Cytodex beads radiation force,
gravity/buoyancy force, and Stokes' drag force to particle size.
The horizontal axis is in microns (.mu.m) and the vertical axis is
in Newtons (N).
[0099] FIG. 64 is a graph of viable cell density (VCD) (10.sup.6
cells/mL) over time for specified AWS power (%) and specified
harvest to recirculation ratios (%). The x-axis (culture day) runs
from 0 to 30 days in intervals of 5 days. The left-side y-axis
(VCD) runs from 0 to 80.times.10.sup.6 cells/mL in intervals of 1
before the break and intervals of 20 after the break, and is used
for the circles. The first right-side y-axis (AWS power) runs from
0 to 60 in intervals of 10, and applies to the line that starts at
about 30% and ends at about 10%. The second right-side y-axis
(harvest to recirculation ratio) runs from 0.0 to 0.5 in intervals
of 0.1, and applies to the line that starts at about 0.15% and ends
at 0.3%. A vertical dashed line shows that AWS began on day 7. The
darker line extending from the vertical dash line corresponds to
the AWS power, and the lighter line extending from the vertical
dashed line corresponds to the harvest to recirculation ratio. The
white dots represent the AWS harvest, and the black dots represent
the bioreactor.
[0100] FIG. 65 is a space characterization of the harvest to
recirculation flow ratio (%) versus the AWS power (%). The y-axis
(harvest to recirculation flow ratio) runs from 0.2 to 1.2 in
intervals of 0.2. The x-axis (AWS power) runs from 10 to 60 in
intervals of 10.
[0101] FIGS. 66A-66C are graphs of AWS versus TFF mAb titer and
product transmission. In all three graphs, AWS is diamonds, TFF
with bleed is squares, and TFF without bleed is triangles. FIG. 66A
shows the mAb titer on the y-axis versus the culture day on the
x-axis. The production of the mAb titer is higher for the AWS than
it is for the TFF where the cells are bled away. FIG. 66B is a
graph comparing the transmission of monoclonal antibody (mAb)
versus culture day. The y-axis is % transmission, and the x-axis is
the culture day. The dotted line indicates 100% transmission.
Higher % transmission is better. The mAb transmission is higher for
the AWS versus the TFF with or without cell bleed. FIG. 66C shows
relative mAb residence time on the y-axis versus culture day on the
x-axis. The relative residence time for the mAb separated by AWS is
lower than the relative residence time for the TFF with or without
cell bleed.
[0102] FIG. 67 is a graph of cell separation efficiency over time
for AWS cell separation at a 10 L scale. The x-axis (culture day)
runs from 0 to 16 days in intervals of 2 days, and is used for the
middle line that starts at x=0 days. The first left-side y-axis
(harvest VCD) runs from 0 to 8.times.10.sup.6 cells/mL in intervals
of 2, and is used for the bottom line that starts at roughly x=1
day. The second left-side y-axis (bioreactor VCD) runs from 0 to
80.times.10.sup.6 cells/mL in intervals of 20. The right-side
y-axis (cell separation efficiency) runs from 0.0 to 1.0 in
intervals of 0.2, and is used for the top line. The cell separation
efficiency is a percentage (i.e., 0.2 corresponds to 20%
efficiency, 0.6 corresponds to 60% efficiency, 1.0 corresponds to
100% efficiency, etc.)
[0103] FIG. 68A is a graph showing the relative abundance of high
molecular weight (HMW) species, monomer species, and low molecular
weight (LMW) species of a monoclonal antibody (mAb) after size
exclusion chromatography (SEC). The darker bars represent the
bioreactor, and the lighter bars represent the harvest. FIG. 68B is
a graph showing the relative abundance of acidic, main peak, and
basic species of a mAb after ion exchange chromatography (IEX).
Again, the darker bars represent the bioreactor, and the lighter
bars represent the harvest. FIG. 68C is a bar graph showing
glycosylation of proteins, showing the proteins sent through the
acoustic perfusion device do not differ from proteins sent through
a TFF device. BRX refers to the bioreactor, and HARV indicates the
harvested material.
[0104] FIGS. 69A-69B are graphs comparing the AWS process to a
tangential flow filtration (TFF) perfusion process using a 0.22
micron membrane. The darker bars represent AWS and the lighter bars
represent TFF. FIG. 69A shows the relative abundance of HMW
species, monomer species, and LMW species of a mAb after size
exclusion chromatography (SEC). FIG. 69B shows the relative
abundance of acidic, main peak, and basic species of a mAb after
IEX.
[0105] FIG. 70 is a perspective view of an eighth exemplary
embodiment of an acoustic perfusion device of the present
disclosure.
DETAILED DESCRIPTION
[0106] The present disclosure may be understood more readily by
reference to the following detailed description of desired
embodiments and the examples included therein. In the following
specification and the claims which follow, reference will be made
to a number of terms which shall be defined to have the following
meanings.
[0107] Although specific terms are used in the following
description for the sake of clarity, these terms are intended to
refer to the particular structure of the embodiments selected for
illustration in the drawings, and are not intended to define or
limit the scope of the disclosure. In the drawings and the
following description below, it is to be understood that like
numeric designations refer to components of like function.
[0108] The singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise.
[0109] The term "comprising" is used herein as requiring the
presence of the named component and allowing the presence of other
components. The term "comprising" should be construed to include
the term "consisting of", which allows the presence of only the
named component, along with any impurities that might result from
the manufacture of the named component.
[0110] Numerical values should be understood to include numerical
values which are the same when reduced to the same number of
significant figures and numerical values which differ from the
stated value by less than the experimental error of conventional
measurement technique of the type described in the present
application to determine the value.
[0111] All ranges disclosed herein are inclusive of the recited
endpoint and independently combinable (for example, the range of
"from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams
and 10 grams, and all the intermediate values). The endpoints of
the ranges and any values disclosed herein are not limited to the
precise range or value; they are sufficiently imprecise to include
values approximating these ranges and/or values.
[0112] The modifier "about" used in connection with a quantity is
inclusive of the stated value and has the meaning dictated by the
context. When used in the context of a range, the modifier "about"
should also be considered as disclosing the range defined by the
absolute values of the two endpoints. For example, the range of
"from about 2 to about 10" also discloses the range "from 2 to 10."
The term "about" may refer to plus or minus 10% of the indicated
number. For example, "about 10%" may indicate a range of 9% to 11%,
and "about 1" may mean from 0.9-1.1.
[0113] It should be noted that many of the terms used herein are
relative terms. For example, the terms "upper" and "lower" are
relative to each other in location, i.e. an upper component is
located at a higher elevation than a lower component in a given
orientation, but these terms can change if the device is flipped.
The terms "inlet" and "outlet" are relative to a fluid flowing
through them with respect to a given structure, e.g. a fluid flows
through the inlet into the structure and flows through the outlet
out of the structure. The terms "upstream" and "downstream" are
relative to the direction in which a fluid flows through various
components, i.e. the flow fluids through an upstream component
prior to flowing through the downstream component. It should be
noted that in a loop, a first component can be described as being
both upstream of and downstream of a second component.
[0114] The terms "horizontal" and "vertical" are used to indicate
direction relative to an absolute reference, i.e. ground level.
However, these terms should not be construed to require structures
to be absolutely parallel or absolutely perpendicular to each
other. For example, a first vertical structure and a second
vertical structure are not necessarily parallel to each other. The
terms "top" and "bottom" or "base" are used to refer to surfaces
where the top is always higher than the bottom/base relative to an
absolute reference, i.e. the surface of the earth. The terms
"upwards" and "downwards" are also relative to an absolute
reference; upwards is always against the gravity of the earth.
[0115] The present application refers to "the same order of
magnitude." Two numbers are of the same order of magnitude if the
quotient of the larger number divided by the smaller number is a
value of at least 1 and less than 10.
[0116] The term "beads" is used herein to refer to microspheres,
microbeads, nano beads, microcarriers and other micro and nano
particulates that are in the micrometer and nanometer size range,
which may be functionalized or non-functionalized, and are composed
of any one or more of a variety of materials and may be of one or
more various shapes.
[0117] Bioreactors are useful for making biomolecules such as
recombinant proteins or monoclonal antibodies. Very generally,
cells are cultured in a bioreactor vessel with media in order to
produce the desired product, and the desired product is then
harvested by separation from the cells and media in an acoustic
perfusion device, such as the device of the present disclosure. The
acoustic filtering device permits the withdrawal of some desired
product, a small portion of the media, and cellular
fragments/debris smaller than the cells, with the remainder being
recycled back to the bioreactor (particularly the cells). The use
of mammalian cell cultures including Chinese hamster ovary (CHO),
NSO hybridoma cells, baby hamster kidney (BHK) cells, insect cells,
and human cells (e.g. T-cells, B-cells, stem cells, red blood
cells), and living/biological cells in general has proven to be a
very efficacious way of producing/expressing the recombinant
proteins and monoclonal antibodies used in various applications
such as pharmaceuticals or vaccines. Two general types of
bioreactor processes exist: fed-batch and perfusion.
[0118] While fed-batch reactors are the norm currently, due mainly
to the familiarity of the process to many scientists and
technicians, perfusion technology is growing at a very fast rate.
Many factors favor the use of a perfusion bioreactor process,
primarily because it is conducive to continuous production. The
capital and start-up costs for perfusion bioreactors are lower,
there is a smaller demand on upstream and downstream capacity,
throughput can be higher, the process is continuous, and the
process uses smaller volumes and fewer seed steps than fed-batch
methods. A perfusion bioreactor process also lends itself better to
development, scale-up, optimization, parameter sensitivity studies,
and validation.
[0119] A perfusion bioreactor may also be utilized to generate
cells that would be utilized in a cell therapy process. In this
type of perfusion bioreactor, biological cells such as CAR T-cells,
Jurkat T-cells and the like are cultured in a perfusion bioreactor.
The acoustic standing wave used in the perfusion devices of the
present disclosure can be used to separate viable and nonviable
cells after the transfection process. This separation ability
allows for improved efficacy of the inoculation of the patient with
this T-cell therapy, since viable cells may be primarily utilized.
The nonviable cells and cell fragments are separated out through
the perfusion process, with these materials going into the
secondary flow and exiting the bioreactor.
[0120] A perfusion bioreactor may also be used for production of
exosomes, microvesicles, or vesicles by cells. The acoustic
perfusion device can then be used to harvest the exosomes, or other
desired cell products, or other targets in a host fluid. In a
similar fashion, a perfusion bioreactor can be used to produce
viruses, such as lentivirus, which are used in cell and gene
therapy to transfect cells. The acoustic perfusion device can then
be used to harvest the virus. In all cases, the device is a cell
retention device.
[0121] Recent developments in perfusion bioreactor technology also
favor its use. Control technology and general support equipment is
improving for perfusion bioreactors, increasing the robustness of
perfusion processes. The perfusion process can now be scaled up to
bioreactors having a volume up to 1000 liters (L). Better cell
retention systems for perfusion bioreactors result in lower cell
loss and greater cell densities than have been seen previously.
Cell densities greater than 50 million cells/mL are now achievable,
compared to fed-batch cell densities of around 20 million cells/mL.
Lower contamination and infection rates have improved the output of
perfusion bioreactors. Higher product concentrations in the harvest
and better yields without significant increase in cost have thus
resulted for perfusion processes.
[0122] Perfusion bioreactors are particularly attractive because of
the continuous production of the biomolecules from the expressing
cell culture, and shorter residence time of said biomolecules in
the process prior to harvest. The target cells are held back by a
filtration process, such as tangential flow filtration (TFF) or
alternating tangential flow filtration (ATF) while the expressed
biomolecules are extracted from the perfusion bioreactor. The cells
are returned to the bioreactor to receive the nutrition and oxygen
to maintain the production of the overall cell culture. In the
perfusion reactor process, the cells continue to multiply, and some
cells may be removed or bled off from the cell culture population
throughout the perfusion production process.
[0123] The TFF and ATF processes of filtration have several issues,
such as clogging/fouling and loss of biomolecule product
(particularly at high cell densities), all directly related to the
nature of the hollow fiber membranes used in the filtration. It is
therefore desirable to find a new filtration process that does not
clog and minimizes loss of the desired biomolecule product. In
addition, TFF and ATF will retain all cellular debris and fines
within the bioreactor, which is not desirable. A process capable of
distinguishing between cell retention while allowing for the
passing of cell debris and fines may therefore be favorable.
[0124] Briefly, the present disclosure relates to acoustic
perfusion devices capable of generating multi-dimensional acoustic
standing wave(s) from one or more piezoelectric transducers, where
the transducers are electrically excited such that they move in a
multimode displacement pattern rather than a "piston" mode of
vibration. Through this manner of acoustic standing wave
generation, a higher lateral trapping force is generated than if
the piezoelectric transducer is excited in a "piston" mode where
one large standing wave is generated. Thus, with the same input
power to a piezoelectric transducer, the multi-dimensional acoustic
standing waves can have a higher lateral trapping force compared to
a planar acoustic standing wave. The input power is tunable for a
controlled flow. This tunability can be used to facilitate
proteinaceous fluid purification of a fluid stream coming from a
bioreactor. Alternatively, the acoustic standing wave may also be a
planar standing wave where the piezoelectric transducer is excited
in the piston mode, generating a planar wave. The acoustic standing
wave(s) may also be a combination of planar and multi-dimensional
acoustic standing waves. All of these standing waves generate an
"interface effect" such that the cells from the bioreactor are held
back and the biomolecule product expressed from the cells, cell
fragments and small debris are allowed to pass through.
[0125] Acoustophoresis is a low-power, no-pressure-drop, no-clog,
solid-state approach to particle separation from fluid dispersions
(i.e., it is used to achieve separations that are more typically
performed with porous filters, but it has none of the disadvantages
of filters). In particular, the acoustic perfusion devices of the
present disclosure are suitable for use with macro-scale
bioreactors for separations in flowing systems with high flow
rates. The acoustic perfusion device is designed to create a high
intensity multi-dimensional ultrasonic standing wave that results
in an acoustic radiation force that can overcome the combined
effects of fluid drag and buoyancy or gravity at certain flow
rates. As a result, the radiation force acts as a filter that
prevents targeted particles (e.g., biological cells) from crossing
through the standing wave. As explained above, the trapping
capability of a standing wave may be varied as desired, for example
by varying the flow rate of the fluid, the acoustic radiation
force, and the shape of the acoustic filtering device to maximize
cell retention through trapping and settling. This technology
offers a green and sustainable alternative for separation of
secondary phases with a significant reduction in cost of energy.
Excellent particle separation efficiencies have been demonstrated
for particle sizes as small as one micron.
[0126] Generally, an acoustic standing wave generates pressure
minima at locations on the standing wave where the amplitude is
minimum and maximum. These are called, respectively, nodes and
anti-nodes. These pressure minima nodes and anti-nodes may be
utilized to capture materials that are differentiated from the
surrounding environment by size, density and compressibility (i.e.,
the speed of sound through the material). Those materials that
collect at the pressure minima nodes are known as having a positive
contrast factor. Those materials that collect at the pressure
minima anti-nodes are known as having a negative contrast
factor.
[0127] In a typical experiment, the system is driven such that the
particles are trapped in the ultrasonic standing wave, i.e., remain
in a stationary position. The axial component of the acoustic
radiation force drives the particles, with a positive contrast
factor, to the pressure nodal planes, whereas particles with a
negative contrast factor are driven to the pressure anti-nodal
planes. The radial or lateral component of the acoustic radiation
force is the force that contributes to trapping the particle. The
forces acting on the particle may be greater than the combined
effect of fluid drag force and gravitational force.
[0128] Generally, the scattering of the acoustic field off the
particles results in a three-dimensional acoustic radiation force,
which acts as a three-dimensional trapping field. The acoustic
radiation force is proportional to the particle volume (e.g., the
cube of the radius) when the particle is small relative to the
wavelength. The force is proportional to frequency and the acoustic
contrast factor. The force also scales with acoustic energy (e.g.,
the square of the acoustic pressure amplitude). For harmonic
excitation, the sinusoidal spatial variation of the force is what
drives the particles to the stable positions within the standing
waves. When the acoustic radiation force exerted on the particles
is stronger than the combined effect of fluid drag force and
buoyancy/gravitational force, the particle is trapped within the
acoustic standing wave field. The action of the lateral and axial
acoustic forces on the trapped particles results in formation of
tightly packed clusters through concentration, clustering,
clumping, agglomeration and/or coalescence of particles that, when
reaching a critical size, settle continuously through enhanced
gravity for particles heavier than the host fluid or rise out
through enhanced buoyancy for particles lighter than the host
fluid. Additionally, secondary inter-particle forces, such as
Bjerkness forces, aid in particle agglomeration. Relatively large
solids of one material can thus be separated from smaller particles
of a different material, the same material, and/or the host fluid
through enhanced gravitational/buoyancy separation.
[0129] Most biological cell types present a higher density and
lower compressibility than the medium in which they are suspended,
so that the acoustic contrast factor between the cells and the
medium has a positive value. As a result, the axial acoustic
radiation force (ARF) drives the cells towards the standing wave
pressure nodes. The axial component of the acoustic radiation force
drives the cells, with a positive contrast factor, to the pressure
nodes, whereas cells or other particles with a negative contrast
factor are driven to the pressure anti-nodes. The radial or lateral
component of the acoustic radiation force is the force that traps
the cells. The radial or lateral component of the ARF is larger
than the combined effect of fluid drag force and gravitational
force. For small particles or emulsions, the drag force FD and
Stokes' drag F.sub.SD can be expressed as:
F D = 4 .pi. .mu. f R P ( U f - U p ) [ 1 + 3 2 .mu. ^ 1 + .mu. ^ ]
, F S D = 6 .pi. .mu. f R P V s .fwdarw. ( 1 ) ##EQU00001##
where U.sub.f and U.sub.p are the fluid and particle velocity,
R.sub.p is the particle radius, .mu..sub.f and .mu..sub.p are the
dynamic viscosity of the fluid and particle, respectively,
{circumflex over (.mu.)}=.mu..sub.p/.mu..sub.f is the ratio of
dynamic viscosities, and {right arrow over (V.sub.s)} is the
particle settling velocity given by:
V S = 2 ( .rho. p - .rho. f ) g R P 2 9 .mu. f ( 2 )
##EQU00002##
where .rho..sub.f is the fluid density, .rho..sub.p is the particle
density, and g is the universal gravitational constant.
[0130] The gravity/buoyancy force FB is expressed as:
F B = 4 3 .pi. R P 3 g ( .rho. f - .rho. p ) ( 3 ) ##EQU00003##
[0131] For a particle to be trapped in the ultrasonic standing wave
and develop into a particle cluster, the force balance on the
particle can be assumed to be zero, and therefore an expression for
lateral acoustic radiation force F.sub.LRF can be found, which is
given by:
F.sub.LRF=F.sub.D+F.sub.B (4)
[0132] For a particle of known size and material property, and for
a given flow rate, this equation can be used to estimate the
magnitude of the lateral acoustic radiation force.
[0133] The theoretical model that is used to calculate the acoustic
radiation force is the formulation developed by Gor'kov, where the
primary acoustic radiation force F.sub.A is defined as a function
of a field potential U, F.sub.A=-.gradient.(U), where the field
potential U is defined as
U = V O [ p 2 ( x , y , z ) 2 .rho. f c f 2 f 1 - 3 .rho. f v 2 ( x
, y , z ) 4 f 2 ] ( 5 ) ##EQU00004##
and f.sub.1 and f.sub.2 are the monopole and dipole contributions
defined by
f 1 = 1 - 1 .LAMBDA. .sigma. 2 f 2 = 2 ( .LAMBDA. - 1 ) 2 .LAMBDA.
+ 1 ( 6 ) ##EQU00005##
where
.sigma. = c p c f .LAMBDA. = p p p f .beta. f = 1 .rho. f c f 2 ( 7
) ##EQU00006##
where p is the acoustic pressure, u is the fluid particle velocity,
.LAMBDA. is the ratio of cell density .rho..sub.p to fluid density
.rho..sub.f, .sigma. is the ratio of cell sound speed c.sub.p to
fluid sound speed c.sub.f, V.sub.o=.pi.R.sub.p.sup.3 is the volume
of the cell, and < > indicates time averaging over the period
of the wave.
[0134] For a one dimensional standing wave, the acoustic pressure
is expressed as
p=A cos(kx)cos(.omega.t) (8)
where A is the acoustic pressure amplitude, k is the wavenumber,
and .omega. is the angular frequency. In this case, the axial
component of the acoustic radiation force F.sub.ARF, is found to
be
F A R F = V O k X A 2 4 .rho. f c f 2 sin ( 2 k x ) ( 9 )
##EQU00007##
where X is the contrast factor given by
X = ( 5 .LAMBDA. - 2 1 + 2 .LAMBDA. - 1 .sigma. 2 .LAMBDA. ) ( 10 )
##EQU00008##
[0135] Particles with a positive contrast factor will be driven to
the pressure nodal planes, and particles with a negative contrast
factor will be driven to the pressure anti-nodal planes. In this
way, the generation of a multi-dimensional acoustic standing wave
in a flow chamber results in the creation of tightly packed
clusters of particles in the flow chamber, typically corresponding
to the location of the pressure nodes or anti-nodes in the standing
wave depending on acoustic contrast factor.
[0136] Gork'ov's model is for a single particle in a standing wave
and is limited to particle sizes that are small with respect to the
wavelength of the sound fields in the fluid and the particle. It
also does not take into account the effect of viscosity of the
fluid and the particle on the radiation force. As a result, this
model cannot be used for the macro-scale ultrasonic separators
discussed herein since particle clusters can grow quite large. A
more complex and complete model for acoustic radiation forces
without any restriction as to particle size relative to wavelength
was therefore used. The models that were implemented are based on
the theoretical work of Yurii Ilinskii and Evgenia Zabolotskaya as
described in AIP Conference Proceedings, Vol. 1474-1, pp. 255-258
(2012) and "Acoustic radiation force of a sphere without
restriction to axisymmetric fields," Proceedings of Meetings on
Acoustics, Vol. 19, 045004 (2013). These models also include the
effect of fluid and particle viscosity, and therefore are a more
accurate calculation of the acoustic radiation force. With these
models, the acoustic radiation force is defined by
F .fwdarw. A z = i 4 K k 2 n = 0 n max m = - n n ( n + m + 1 ) ( n
- m + 1 ) ( 2 n + 1 ) ( 2 n + 3 ) [ A n * + A n + 1 + 2 A n * A n +
1 ] a n , m * a ( n + 1 , m ) + c . c . ( 11 ) ##EQU00009##
where a.sub.n,m* and a.sub.(n+1,m) are the expansion coefficients
of external field potential with respect to spherical functions
j.sub.n(kr)Y.sub.n,m(.theta.,.PHI.), Y.sub.n,m(.theta.,.PHI.) are
spherical harmonies, A.sub.n is the scattering coefficient for mode
(m,n), K is bulk modulus, and k is the wavenumber in the
liquid.
[0137] Cells that have a low contrast factor compared to the fluid
in which they are transported are more difficult to separate using
an acoustic standing wave. Through specialized perturbations of a
piezoelectric material, higher order modes of vibration in the
piezoelectric material may be generated. When this piezoelectric
material that is perturbed in a multimode fashion is coupled with a
reflector, a specialized type of acoustic standing wave, known as a
multi-dimensional acoustic standing wave, is generated. In this
way, target biological cells having low cell concentrations (e.g.,
T cells) may be separated from a fluid medium utilizing a
multi-dimensional acoustic standing wave. The target biological
cells are generally at lower concentrations than, for example, a
CHO cell population with 30 million cells per mL versus a
concentration of 1 million cells per mL for Jurkat T cells. Thus,
the low contrast cells, such as Jurkat T cells, in a low population
concentration are separated continuously from the fluid media
within which they are entrained by utilizing a multi-dimensional
acoustic standing wave.
[0138] Desirably, the ultrasonic transducer(s) generates a
multi-dimensional standing wave in the fluid that exerts a lateral
force on the suspended particles to accompany the axial force. The
multi-dimensional standing wave generates acoustic radiation forces
in both the axial direction (e.g., in the direction of the standing
wave, between the transducer and the reflector, which may be at an
angle across the flow direction, and in some instances may be
perpendicular to the flow direction) and the lateral direction
(e.g., in the flow direction or transverse to the direction between
the transducer and the reflector). As the mixture flows through the
acoustic chamber, particles in suspension experience a strong axial
force component in the direction of the standing wave. Since this
acoustic force is across (e.g. perpendicular to) the flow direction
and the drag force, it quickly moves the particles to pressure
nodal planes or anti-nodal planes, depending on the contrast factor
of the particle. The lateral acoustic radiation force acts to move
the concentrated particles towards the center of each planar node,
resulting in clustering, agglomeration or clumping. The lateral
acoustic radiation force component can overcome fluid drag for such
clumps of particles, to continually grow the clusters, which can
exit the mixture due to gravity or buoyancy. The drop in drag per
particle as the particle cluster increases in size, as well as the
drop in acoustic radiation force per particle as the particle
cluster grows in size, may separately or collectively influence
operation of the acoustic separator device. In the present
disclosure, the lateral force component and the axial force
component of the multi-dimensional acoustic standing wave are of
the same or different order of magnitude. In some particular
embodiments, the ratio of the lateral force component to the axial
force component is about 0.5 or less. In this regard, it is noted
that in a multi-dimensional acoustic standing wave generated by a
single transducer, the axial force is stronger than the lateral
force, but the lateral force of such a multi-dimensional acoustic
standing wave is much higher than the lateral force of a planar
standing wave, usually by two orders of magnitude or more. Typical
results published in literature state that the lateral force is two
orders of magnitude smaller than the axial force. In contrast, the
technology disclosed in this application provides for a lateral
force to be of the same order of magnitude as the axial force.
However, in certain embodiments described further herein, the
device use both transducers that produce multi-dimensional acoustic
standing waves and transducers that produce planar acoustic
standing waves. For purposes of this disclosure, a standing wave
where the lateral force is not the same order of magnitude as the
axial force is considered a "planar acoustic standing wave." The
lateral force component of the total acoustic radiation force (ARF)
generated by the ultrasonic transducer(s) of the present disclosure
is significant and is sufficient to overcome the fluid drag force
at linear velocities of up to 1 cm/s, and to create tightly packed
clusters, and is of the same order of magnitude as the axial force
component of the total acoustic radiation force.
[0139] The average size of microvesicles, such as exosomes (i.e.,
about 150 nanometers in diameter or smaller), makes it difficult to
separate the same using normal acoustophoretic techniques. Through
the use of beads, the acoustic footprint or contrast factor of the
exosomes may be increased dramatically by functionalizing the
surface of the bead such that it will attach to the exosomes and
create a moiety that is subject to acoustophoresis through its high
contrast factor. The beads may have either a positive or negative
contrast factor. Positive contrast factor materials include
polystyrene beads, glass beads and other materials whose density is
greater than that of the host fluid that contains the exosomes.
Negative contrast factor materials of interest include
microbubbles, micro-glass spheres and other low-density beads that
may be functionalized to attract exosomes and thus increase their
contrast factor footprint. The functionalization of the beads may
be by, for example, attachment of CD 9, CD 63, CD 81, or EP
CAM.
[0140] The acoustophoresis separation processes of the present
disclosure may be coupled with other techniques, such as field flow
fractionation where the ultrasonic wave is utilized to fractionate
the materials entrained in a fluid stream, such as the exosome
moieties or the beads that are carrying the exosomes through
affinity binding. The acoustophoresis separation processes of the
present disclosure may also be coupled with tangential flow
filtration in a flow field that is bounded by the acoustic standing
wave.
[0141] In the case of exosomal separation, an efficient and cost
effective platform for complete isolation and segregation of
extracellular vesicle populations from cell culturing, without the
potential for lysis to occur, is possible with the acoustic
tangential flow filtration processes described herein, whereas the
detrimental processes of rupture and deactivation would happen with
physical filtration and/or centrifugation. With emphasis on
yielding pure exosome or oncosome populations that are
morphologically and functionally intact, the acoustic tangential
flow filtration process offers a great improvement. The acoustic
tangential flow filtration process technology is readily adapted to
enable automated workflows and reduce human intervention, enable
closed loop systems, and to obtain the enriched distinct
preparations of exosomes and oncosomes used to provide diagnostic
determinations and selective therapeutic product.
[0142] It may be helpful to contrast the technology of the present
disclosure with that of prior filtration technology. FIG. 40 shows
two prior art filtration methods. The left-hand side of FIG. 40
illustrates direct flow filtration (DFF). In DFF, the entire feed
stream 4005 of fluid and particles is directed towards the filter.
The filter 4010 holds back the particles 4020 that are larger than
the filter's pore size, whereas smaller particles 4030 and the
fluid pass through the filter. The right-hand side of FIG. 40
illustrates tangential flow filtration (TFF). In TFF, the feed
stream is not directed towards the filter. Rather, the feed stream
is directed tangentially to the filter, such that a majority of the
feed stream passes tangentially over the filter surface. Typically,
this feed stream is recirculated to pass by the filter more than
once. A much smaller filtrate stream 4006 is pulled through the
filter membrane containing the smaller particles 4030. One
advantage of TFF over DFF is that the tangential stream reduces the
clogging and fouling of the filter and the formation of a gel layer
that sits on top of the filter.
[0143] In the devices of the present disclosure, during startup,
the fluid ensonified by the acoustic standing wave is clarified by
the process of trapping cells and growing them into tightly packed
clusters, such that continuous gravitational separation of the
clusters of cells takes place. Since there is a limited amount of
new cells flowing into this volume, this results in a rapid
clarification of the fluid subjected to the acoustic standing wave.
When this state is reached, the system can be described as
including two fluids: a first fluid containing the desired product
and small cell fragments/debris (which have passed through the
acoustic standing wave), and a second fluid containing the
bioreactor fluid and all of the cells (which are held back by the
acoustic standing wave). The two fluids may be of different
effective acoustic properties, such as density and speed of sound,
with a well-defined interface between these two fluids. The
acoustic standing wave is a three-dimensional acoustic field,
which, in the case of excitation by a rectangular transducer, can
be described as occupying a roughly rectangular prism volume of
fluid. Typically, two opposing faces are the transducer and
reflector, an adjacent pair of opposing faces are the walls of the
device, and the final opposing pair of faces, the upstream and
downstream faces of the cube, extend through the fluid. The
interface between the two fluids is generally located near the
upstream face of the acoustic standing wave field, generating an
"acoustic barrier or edge effect". This location is also referred
to as an upstream interface region. The first fluid (i.e., the
fluid that has been clarified and contains the product, some cells,
and cell fragments) is downstream of the interface and represents
the harvest flow and occupies the volume of fluid ensonified by the
acoustic standing wave field. The second fluid (i.e., the fluid
containing the bioreactor fluid and most of the cells) is upstream
of the interface. These two different fluids can be seen in the
photo on the right in FIG. 33. During operation at increased flow
rates, the interface effect location may move downstream and is
then located within the volume of fluid ensonified by the
transducer.
[0144] The acoustic standing wave field exerts an acoustic
radiation pressure (i.e. a pressure rise) and an acoustic radiation
force on the cells at the interface region between the two fluids,
thereby keeping the upstream cells from entering the acoustic
field. The occurrence of the radiation pressure and the force on
the interface allows for the first fluid containing the product to
pass through the interface while retaining the cells in the
upstream fluid. The cells that are held back by the effect of the
acoustic radiation force at the interface between the two fluids
can be continuously returned to the bioreactor to ensure they
receive the nutrition and oxygen to maintain the production of the
overall cell culture.
[0145] The circulating motion of the flow field underneath the
interface transports the cells that are retained by the acoustic
field back to the bioreactor. The circulating flow motion is driven
by the primary recirculation stream and can be optimized with
acoustic chamber geometry variations for maximum system efficiency.
This process or configuration is discussed further below with
respect to FIG. 33.
[0146] During perfusion, the acoustic perfusion devices of the
present disclosure have multiple possible modes of operation. One
of these modes may be dominant in the device or they may occur
concurrently depending on the distribution of cells and fluid
within the device. In a first mode of operation illustrated in FIG.
41 (Mode 1), the fluid containing larger particles 4020 (light
color) enters the acoustic standing wave field 4040, which is
produced here between transducer 4008 and reflector 4009. A
multi-dimensional acoustic standing wave traps the particles at
specific points, packs the particles into tightly packed clusters
4022, and continuously separates the clusters through enhanced
gravitational settling. The particle clusters settle out, enter the
tangential flow path (indicated by arrow 4001) and are redirected
to the bioreactor by the recirculation stream. Smaller particles
4030 (darker color) are not trapped by, and pass through, the
acoustic standing wave, to be harvested. The harvest flow direction
is indicated by arrow 4002. The orientation of this device is
significant, and the direction of gravity is also indicated. In
particular embodiments, the larger particles 4020 can be cells and
the smaller particles 4030 can be cell debris and/or fines. In
other embodiments, the larger particles 4020 can be beads and the
smaller particles 4030 can be cells or exosomes (or more generally
the desired target material). In other embodiments, the larger
particles 4020 can be beads with attached antibodies and the
smaller particles 4030 can be cells that are not targeted by the
functionalized beads.
[0147] The second mode of operation (Mode 2) is illustrated in FIG.
42, where the acoustophoretic system creates a strong barrier for
cells at the interface between the two fluids and prevents cells
from entering the acoustic field. Here, a barrier of cells is
established between the two fluids through the interface effect of
the acoustic standing wave. A first clarified fluid stream 4050
contains the smaller particles/desired byproducts 4030 within the
acoustic standing wave field and the harvest stream. A second fluid
stream 4055 contains the retained cells 4020 upstream of the
acoustic standing wave field. The harvest flow direction is
indicated by arrow 4002. In this mode of operation, an acoustic
interface effect is realized, as indicated by dotted line 4007
(representing the interface region between the two fluids,
clarified fluid downstream and flow mixture and cells on the
upstream side). Very generally, the acoustic interface effect holds
the cells back and prevents them from entering the acoustic field
while a portion of the fluid stream containing the produced
biomolecules and cell fragments is permitted to pass through this
barrier. The tangential flow path underneath the acoustic interface
(arrow 4001) collects the retained cells and flows them back into
the main recirculation stream and back to the bioreactor. This
recirculation is discussed further below with respect to FIG. 32
and FIG. 61. Again, the direction of gravity is indicated in the
figures. Alternatively, the second fluid stream 4055 can contain
desired target material which is retained within the perfusion
device, and the first fluid stream 4050 can be returned to the
bioreactor.
[0148] In perfusion applications, the setup of the acoustophoretic
device is similar to that of TFF. A feed stream containing the
cells, cell debris, fines, and product, i.e., protein, flows from
the bioreactor into the perfusion system. A portion of the stream
flows in a tangential fashion along the upstream/lower interface
region of the acoustic standing wave and is recirculated back to
the bioreactor. A smaller portion of the feed stream is harvested,
i.e., diverted and flows through the acoustic standing wave. Here
the acoustic standing wave functions very similarly to the filter
in TFF, preventing the cells from entering the acoustic field. The
harvest stream contains smaller particles such as cell debris and
fines as well as the desired biomolecule product. The cells that
are retained by the acoustic standing wave are transported by the
recirculation stream back to the bioreactor. FIG. 32 and FIG. 61,
which are discussed further herein, also illustrate perfusion
devices that use a tangential flow stream.
[0149] Perfusion applications typically entail high cell densities,
e.g., >50 million cells/mL, and lower harvest velocities
contrary to cell clarification or oil/water applications. The two
fluid streams also have different effective acoustic properties,
i.e., speed of sound and density of the media/cell mixture. As cell
density increases, the difference in acoustic properties of the two
fluid streams are more pronounced. The acoustic standing wave field
exerts an acoustic radiation pressure, i.e., a pressure rise, on
the second fluid stream, enriched with cells, as well as acoustic
radiation forces on the cells suspended in the fluid. This
radiation pressure and radiation force act at the interface between
the two fluids which coincides with the upstream bounding surface
of the acoustic field. When this "acoustic interface" effect of
acoustic radiation force is sufficiently strong, it can prevent the
cells from entering the acoustic field. Equally important is a
tangential flow path to collect the retained cells and transport
them back to the bioreactor.
[0150] The acoustic interface effect may also be referred to as an
acoustic wall effect and results from the interface of the acoustic
field exerting a strong lateral force (i.e., in the opposite
direction to the harvest flow and perpendicular to the axis of the
acoustic standing wave) on the suspended particles, thereby keeping
the relatively larger sized particles from entering the acoustic
field and allowing clarified fluid (i.e., the fluid containing the
smaller-sized product) to enter the acoustic field, thereby
creating an acoustic perfusion cell retention device. In this way,
the clarified fluid can escape and the cells are held down by the
radiation force. This force is never positive, meaning that it
always holds the cells down at the interface, i.e., the force is
acting in the upstream flow direction, not allowing the cells to
pass through the acoustic interface. The multiple peaks in the
power curve (see discussion of FIG. 36 below) show the existence of
multiple modes of operation including planar resonance modes and
multi-dimensional modes of operation, indicating that this type of
operation can be generated through utilization of planar and
multi-dimensional standing waves alike. In systems having
1''.times.1'' dimensions, there exists a planar resonance about
every 30 kHz. FIG. 36 shows evidence of additional peaks indicating
the existence of the multi-dimensional modes. Per unit power, these
modes can be equally or even more effective as the planar resonance
modes. As explained above, the cells that are held back by the
acoustic radiation force may then picked up by the scrubbing motion
of the fluid flow field (i.e., the recirculating flow underneath
the interface), and be continuously returned to the bioreactor to
ensure they receive the nutrition and oxygen to maintain the
production of the overall cell culture.
[0151] The clarified fluid contains both the desired products and
cell fragments, all of which are smaller than whole viable cells.
In this way, the media that is returned to the bioreactor is
clarified of cell fragments. Cell fragments absorb media without
producing desired product, making the perfusion process less
efficient. Thus, there is an efficiency gain and a cost savings
obtained by removing these cell fragments using the acoustic
perfusion devices of the present disclosure. Further clarification
of the clarified fluid may be achieved downstream using a second
device or a secondary flow chamber that contains another
transducer-reflector pair that operates at a different frequency.
This arrangement traps, clumps, clusters, or agglomerates particles
having a size of about 10 microns or less that may have passed
through the original acoustic standing wave, in the same manner as
described before. A third transducer-reflector pair operating at
another frequency, 3 MHz to 20 MHz, or higher, may be utilized to
trap, clump, cluster, or agglomerate and drop out the small cell
fragments and debris that passed through the initial acoustic
standing wave and the "interface effect". This triple-clarified
fluid containing the desired biomolecules can then directly enter a
sterile filter. For example, the original acoustic perfusion device
may operate at frequencies up to about 4 MHz. It is contemplated
that the frequency of this second and third acoustic standing wave
field would be from about 6 MHz to about 20 MHz, and possibly
higher, to trap smaller sized cell fragments.
[0152] During startup of a bioreactor at low cell density, e.g., 2
million cells/mL, the first described mode of operation dominates
(FIG. 33, left image). As cell density in the bioreactor increases
over time, the mode of operation gradually switches from mode 1 to
mode 2, and both modes may coexist at the same time.
[0153] When an acoustic standing wave is employed for perfusion in
a bioreactor with an already high cell density, e.g., 50 million
cells/mL, the device typically starts in the first mode of
operation (FIG. 33, left image), until the volume of fluid within
the acoustic standing wave is clarified, at which point the
operation gradually switches to the second described mode of
operation (FIG. 33, right image). At times, during operation, an
instability, usually manifested as a perturbation or oscillation of
the interface between the two fluids, may grow sufficiently strong
such that cells enter the volume of fluid within the acoustic
standing wave, at which point, for a short period of time, the
device acts in a combined mode of operation, where both modes are
active (i.e., the interface effect prevents cells from entering the
acoustic field as explained above, while the acoustic field
clarifies the cells that have entered the volume of fluid within
the acoustic standing wave field). Once the tightly packed cell
clusters have settled out (i.e., once the volume of fluid within
the acoustic standing wave has been sufficiently clarified), the
mode of operation is then again that of the second described mode
of operation, namely, the acoustic interface effect. It is
important to note that the device can operate in both/either of the
modes of operation, as described above, without external switching.
In other words, the properties of the fluid streams, e.g., cell
concentrations in the streams, and acoustic field dictate which
mode dominates.
[0154] The acoustic standing wave(s) perfusion devices of the
present disclosure are operated differently compared to prior
acoustic filter usages, previously described in literature.
Previously, acoustophoresis was operated such that the
protein-producing materials, such as Chinese hamster ovary cells
(CHO cells), the most common host for the industrial production of
recombinant protein therapeutics, were trapped within a planar
ultrasonic standing wave (i.e., remain in a stationary position).
Cells were retained in an acoustic field by causing individual
cells to migrate towards the pressure nodal planes of the planar
acoustic standing wave. There, as the cells were retained in the
standing wave, there was also a physical scrubbing of the cell
culture media flowing past, whereby more cells were trapped as they
came in contact with the cells that were already held within the
standing wave. The standing wave and harvest fluid flow were then
intermittently shut off to allow the cells to drop out of the
standing wave and return to the bioreactor.
[0155] In contrast, in the present disclosure, the ultrasonic
standing waves are used as a blanket or selector or "force field"
in the perfusion device. The perfusion device is configured to
permit fluid flows that are against gravity forces. The flows
against gravity contribute to the biological cells sinking. The
standing wave is created near the top of the filtering device and
acts like a filter to prevent the cells from entering the acoustic
field and exiting through the top of the filtering device (i.e.,
acting similar to a force field holding the cells back from
entering the acoustic field). Thus, two output streams are created,
one output stream retaining the cells and exiting through a port at
the bottom of the device, and the other output stream being
depleted in cells and exiting through a port at the top of the
device (the cell concentration in the two output streams being
compared to each other). In this mode of operation, there is almost
no reliance on clustering, clumping, or agglomeration of the cells
within the acoustic field to achieve separation. The combination of
fluid dynamics and acoustic field to block cells is particularly
advantageous in certain applications. Because the cells are not
retained in the acoustic field for a period of time, the filtering
device is more easily operated in a continuous arrangement.
[0156] Described another way, the acoustic perfusion device has two
fluid streams flowing at different rates. The main fluid stream,
carrying the expressing cell culture, culture media, product, and
other bioreactor constituents, enters the device and is partially
diverted into a secondary, lower volume, lower flow fluid stream.
This secondary fluid stream passes through the multi-dimensional
acoustic standing wave, where the multi-dimensional acoustic
standing wave (or generally the interface effect created by the
acoustic standing wave) holds back the main cell culture and allows
the expressed biomolecules, the monoclonal antibodies and
recombinant proteins, along with other small particles such as
submicron and micron-sized cell debris, to pass through and be
further collected and processed outside/downstream of the
bioreactor. The main fluid stream, containing the main cell
culture, is then recycled back to the bioreactor. The acoustic
standing wave and its "interface effect" can be considered to act
as a filter, preventing large cells, other particles or bodies,
from exiting the bioreactor.
[0157] In another application, the acoustic perfusion devices can
act as a retention device and cell washing device for cell therapy
applications. In continuous cell-culture applications, such as
autologous and allogeneic cell therapy, cells may be initially
harvested at a very low cell-density. The cells may be purified,
isolated and proliferated in the cell culture. Relatively few cells
may be used to seed a bioreactor. The bioreactor may be used to
increase the number of cells in the cell culture. Further
processing steps such as concentrating, washing, and media exchange
are all needed for various applications. The commonality in all
these applications is the need to continuously circulate, add,
and/or remove media while retaining cells in a bioreactor (which
may be traditional or single-use) with no effect to their
viability. The acoustic cell retention systems described herein
operate over a range of cell recirculation rates, efficiently
retain cells over a range of perfusion (or media removal rates),
and can be tuned to fully retain or selectively pass some
percentage of cells through fluid flow rate, transducer power or
frequency manipulation. Power and flow rates can all be monitored
and used as feedback in an automated control system. Specialty flow
paths may also be used such that a small volume of the main fluid
flow is "sipped" off and the expressed biomolecules are separated
from the main cell culture.
[0158] One advantage of acoustophoresis is that the acoustic
radiation force does not harm or negatively affect the biological
cells or the desired biomolecule product. Moreover, perfusion is
continuous, such that the cell culture is kept viable and desired
products can be continually recovered therefrom.
[0159] In a perfusion bioreactor system, it is desirable to be able
to filter and separate the viable biological cells from the
expressed materials that are in the fluid stream (i.e., cell
culture media) and cellular debris. As previously mentioned, such
biological cells may include Chinese hamster ovary (CHO) cells,
whose cell genome is manipulated to express large biomolecules.
Such biomolecules can include recombinant proteins or monoclonal
antibodies, and are the desired product to be recovered.
[0160] The acoustic perfusion devices of the present disclosure are
designed to maintain a high intensity multi-dimensional acoustic
standing wave that can act as a filter, permitting smaller
particles (such as recombinant proteins or cellular debris) to pass
through while excluding larger particles (such as viable cells).
Generally, the device is driven by an oscillator and amplifier (not
shown), and the device performance is monitored and controlled by a
computer (not shown). The parameters of the energy or signal
applied to the transducer, such as, for example, frequency or
voltage amplitude, may be controlled and/or modulated. In some
instances it may be desirable to control the applied signal to
compensate for effects such as acoustic streaming. This control may
be achieved by amplitude modulation and/or by frequency modulation.
The duty cycle of the propagation of the standing wave may also be
utilized to achieve certain results (i.e. the acoustic beam may be
turned on and shut off at different time periods or rates).
[0161] FIG. 63 is a log-log graph (logarithmic y-axis, logarithmic
x-axis) that shows the scaling of the radiation forces of cells
(MRC-5) and microcarriers (Cytodex beads), fluid drag force, and
buoyancy force with particle radius. The graph provides an
explanation for the separation of microvesicles, cells or
microcarriers from a host fluid based on parameter settings. For
example, the frequency, flow rate (drag force) and power can be
controlled and/or modulated to prevent or permit passage of
microvesicles, cells or microcarriers. If the fluid flow is
increased from that illustrated in the graph in FIG. 63, the drag
force intersection with the radiation force curves rises along the
curves and can pass the point where particles the size of Cytodex
beads are repelled, so that microvesicles, cells and beads are all
passing through the acoustic field. At the operating point
illustrated in the graph, cells and microvesicles are passed, while
the beads are repelled. Lowering the fluid flow until the
intersection of the drag force line intersection with the radiation
force curves is below the cell (MRCS) size causes microvesicles
(which are smaller than cells and not shown on the graph) to pass,
while cells and beads are repelled or retained by the acoustic
field. Similar effects can be achieved by modulating and/or
controlling frequency or power of the acoustic field. Thus, the
perfusion systems disclosed herein can discriminate based on size
with a relatively high level of distinction.
[0162] FIG. 1 illustrates a single standing wave system 100 that is
comprised of a reflector plate 101 and an ultrasonic transducer 103
that is set to resonate so as to form a standing wave 102.
Excitation frequencies typically in the range from 100 kHz to 100
MHz are applied by the transducer 103. One or more
multi-dimensional standing waves are created between the transducer
103 and the reflector 101. An ideal standing wave is the sum of two
propagating waves that are equal in frequency and intensity and
that are traveling in opposite directions, i.e. from the transducer
to the reflector and back. The propagating waves constructively
interfere with each other and thus generate the standing wave. A
dotted line 105 is used to indicate the zero-amplitude of the wave.
A node is a point where the wave has minimum amplitude, and is
indicated with reference numeral 107. An anti-node is a point where
the wave has maximum amplitude, and is indicated with reference
numeral 109. The standing wave is illustrated as beginning and
ending with local maxima, however, other implementations are
possible. For example, the standing wave can be offset at the
transducer or the reflector so that local minima or maxima are
spaced from the transducer or from the reflector. The reflected
wave (or wave generated by an opposing transducer) can be in- or
out-of-phase with the transducer generated wave. The
characteristics of the standing wave can be modified and/or
controlled by the drive signal applied to the transducer, such as
by modifying and/or controlling the phase, amplitude, or frequency
of the drive signal. Acoustically transparent or responsive
materials may also be used with the transducer or reflector to
modify and/or control the standing wave.
[0163] FIG. 2 is a schematic diagram that compares a fed-batch
bioreactor system 201 (left side) with a perfusion bioreactor
system 202 (right side). Beginning with the fed-batch bioreactor on
the left, the bioreactor 210 includes a reaction vessel 220. The
cell culture media is fed to the reaction vessel through a feed
inlet 222. An agitator 225 is used to circulate the media
throughout the cell culture. Here, the agitator is depicted as a
set of rotating blades, though any type of system that causes
circulation is contemplated. The bioreactor permits growth of a
seed culture through a growth/production cycle, during which time
debris, waste and unusable cells will accumulate in the bioreactor
and the desired product (e.g. biomolecules such as monoclonal
antibodies, recombinant proteins, hormones, etc.) will be produced
as well. Due to this accumulation, the reaction vessel of a
fed-batch process is typically much larger than that in a perfusion
process. The desired product is then harvested at the end of the
production cycle. The reaction vessel 220 also includes an outlet
224 for removing material.
[0164] Turning now to the perfusion bioreactor 202 on the
right-hand side, again, the bioreactor includes a reaction vessel
220 with a feed inlet 222 for the cell culture media. An agitator
225 is used to circulate the media throughout the cell culture. An
outlet 224 of the reaction vessel is fluidly connected to the inlet
232 of an acoustic perfusion device 230 of the present disclosure,
and continuously feeds the bioreactor contents (containing cells
and desired product) to the filtering device. The perfusion device
is located downstream of the reaction vessel, and separates the
desired product from the cells. The acoustic perfusion device 230
has two separate outlets, a product outlet 234 and a recycle outlet
236. The product outlet 234 fluidly connects the acoustic perfusion
device 230 to a containment vessel 240 downstream of the perfusion
device, which receives the flow of the desired product (plus media)
from the perfusion device. From there, further
processing/purification can occur to isolate/recover the desired
product. For example, further downstream of this acoustic perfusion
device may be additional filters such as an ATF, TFF, depth filter,
centrifuge, etc. The recycle outlet 236 fluidly connects the
acoustic perfusion device 230 back to a recycle inlet 226 of the
reaction vessel 220, and is used to send the cells and cell culture
media back into the reaction vessel for continued
growth/production. Put another way, there is a fluid loop between
the reaction vessel and the perfusion device. The reaction vessel
220 in the perfusion bioreactor system 202 has a continuous
throughput of product and thus can be made smaller. The filtering
process is critical to the throughput of the perfusion bioreactor.
A poor filtering process implies low throughput and results in low
yields of the desired product.
[0165] FIG. 3 is a cross-sectional view of a generic bioreactor 300
that is useful for the systems of the present disclosure. As
illustrated here, the bioreactor includes a reaction vessel 320
having an internal volume 323. A feed inlet 322 at the top of the
vessel is used to feed cell culture media into the vessel. An
agitator 325 is present. An outlet 324 is shown at the bottom of
the vessel. A thermal jacket 310 surrounds the reaction vessel, and
is used to regulate the temperature of the cells/media. An aerator
312 is located on the bottom of the vessel for providing gas to the
internal volume. Sensors 314 are shown at the top right of the
vessel. A pump 316 is illustrated for feeding the cell culture
media into the vessel, as is another pump 318 for removing cell
culture media from the vessel.
[0166] The perfusion systems described above use an acoustic
perfusion device of the present disclosure. The contents of the
bioreactor are continuously flowed through the acoustic perfusion
device to capture the desired products.
[0167] FIG. 4 is a first embodiment of an acoustic perfusion device
400 that can be used with the previously-described systems. The
device includes an inlet port 410, an outlet port 430, a first
collection port 470, a bottom wall 420, and an acoustic chamber
450. The acoustic chamber 450 can also be referred to as a fluid
cell.
[0168] The inlet port 410 is located at a first end 412 of the
device. Generally, the inlet port 410 is fluidly connected to an
associated bioreactor and serves as the inlet through which the
fluid mixture with cells, fines, and product is introduced to the
device. An inlet flow path 451 leads from the inlet port 410 to the
acoustic chamber 450, which contains an internal volume. An upper
wall 411 can be present above the inlet flow path leading from the
inlet port to the acoustic chamber, the upper wall having a
substantially horizontal orientation. The inlet flow path has a
cross-sectional area 452 (illustrated by the dotted square).
[0169] The inlet port 410 is located at a first height 402 above
the outlet port 430, which defines a bottom end of the device. Put
another way, the outlet port 430 is located below the acoustic
chamber 450 or below the inlet port 410, or at the bottom end 416
of the device. The placement of the outlet port 430 below the inlet
port 410 ensures that fluid flow through the device is passively
urged by gravity towards the outlet port 430, and that a hydraulic
head is created within the device. The outlet port 430 may also be
referred to as a fluid recycle port because the host fluid is
recycled or returned from the device to the associated bioreactor
through the outlet port 430. As illustrated here, the outlet port
430 is also located at a second end 414 of the device, opposite the
first end 412. The first end 412 and second end 414 can be
considered as being opposite ends of an x-axis, while the bottom
end 416 and top end 418 can be considered as being opposite ends of
a z-axis.
[0170] The first collection port 470 is located above the acoustic
chamber 450 at the top end 418 of the device, and is fluidly
connected to the acoustic chamber. The device may include
additional collection ports, such as second collection port 472,
which is spaced apart from the first collection port 470. The first
and second collection ports 470, 472 are generally used to harvest
and recover a portion of the desired biomolecule byproducts from
the device. A collection or harvest flow path 453 leads from the
acoustic chamber to the collection ports 470, 472. The collection
flow path has a cross-sectional area 454 (illustrated by the dotted
square). In some particular embodiments, the cross-sectional area
454 of the collection flow path is greater than the cross-sectional
area 452 of the inlet flow path. This arrangement provides one
method by which the flow rate of fluid through the collection ports
470, 472 can be made much lower than the incoming flow rate of
fluid. When used in perfusion biomanufacturing, the collection
ports can also be referred to as perfusion or harvest ports.
Because fluid depleted in cells and enriched in desired biomolecule
products, cell debris, and other fines is harvested, the collection
ports can also be referred to as harvest ports, and the collection
flow path can also be referred to as the harvest flow path.
[0171] In this embodiment, the bottom wall 420 extends from the
inlet port 410 to the outlet port 430 of the device. The exact
shape of the bottom wall 420 can vary to obtain the desired fluid
flow. As illustrated here, the bottom wall 420 curves from the
inlet port 410 to the outlet port 430 of the device. Relative to a
line between the inlet port 410 and the outlet port 430,
illustrated as dotted line 401, the bottom wall 420 has a concave
curve. An outlet flow path 432 leads from the acoustic chamber 450
to the outlet port 430.
[0172] As illustrated here, a first ultrasonic transducer 460 is
located on a sidewall 440 of the device at a second height 404 that
is above the first height 402 (i.e. closer to the top end 418 of
the device) and below the collection ports 470, 472. This volume
above the acoustic chamber 450 and below the collection ports 470,
472 is identified here as a harvest or collection zone 456. The
first ultrasonic transducer 460 includes a piezoelectric material
that can be driven by a drive signal to create a multi-dimensional
standing wave in the acoustic chamber 450 across the collection
flow path 453. An acoustic radiation force field thus separates the
acoustic chamber 450 from the collection ports 470, 472.
[0173] In the embodiment of FIG. 4, the device includes a reflector
480 located on a wall opposite from the first ultrasonic transducer
460. The reflector is also located at the second height (i.e. the
same height as the transducer). Together, the transducer 460 and
reflector 480 generate a multi-dimensional acoustic standing wave,
as illustrated in FIG. 1.
[0174] The inlet port 410, outlet port 430, and the collection
ports 470, 472 are, in this illustrated embodiment, all located on
a front wall 475 of the device. It is also contemplated that these
ports can face in any other direction, as desired. The front wall
475 is illustrated here as having a flat or planar face, and has a
constant thickness. However, the shape of the front wall may also
vary if desired, for example to change the cross-sectional areas
452, 454. Finally, the rear wall of the device is attached to a
mounting piece 490, which contains holes 492 for attaching the
perfusion device to a surface for operation.
[0175] In use, the fluid mixture containing biological cells and
smaller molecules enters the acoustic chamber 450 through the inlet
port 410. Inside the acoustic chamber, gravity acts to drag the
biological cells downwards towards the outlet port 430. A passive
settling process occurs in the acoustic chamber, creating a fluid
with a relatively high concentration of biological cells at the
bottom end 416 of the device, and a fluid with a relatively lower
concentration of biological cells at the top end 418 of the device.
The vast majority of incoming fluid, and thereby, the large
majority of the cell population never passes through the acoustic
standing wave(s). The fluid with the high concentration of
biological cells is pumped back to the bioreactor, and the fluid
with the relatively low concentration of biological cells (and also
containing desired biomolecules) is pumped out and collected though
the collection port(s) 470, 472. The acoustic standing wave(s) of
the device act to prevent significant numbers of biological cells
from exiting through the collection port(s) 470, 472.
[0176] The flow rate through the collection or harvest flow path
453 is, in various embodiments, at least one order of magnitude
smaller than the flow rate through the inlet flow path 451. In more
particular embodiments, the flow rate of the fluid mixture entering
the device through the inlet port is about 1 liter per minute
(L/min) and the flow rate of the fluid depleted in cells exiting
the device through the collection port(s) is about 10 milliliters
per minute (mL/min). In some tests, bioreactors having a size of 2
liters to 10 liters have been tested with solutions containing up
to 10% yeast and up to 50 million cells/mL. The flow rate through
the inlet port has been from about 0.75 L/min to about 3 L/min,
with the flow rate through the collection flow path (i.e. all
collection ports together) being about 1 mL/min to about 30 mL/min.
A 95% cell recovery rate has been achieved.
[0177] The acoustic perfusion devices of the present disclosure can
filter very high cell densities, around 100 million cells per mL
and possibly in the range of about 20 million to about 120 million
cells per mL, whereas other filtering technologies such as ATF may
be limited to being able to filter at densities less than 80
million cells per mL. Unlike hollow fiber membranes, the acoustic
standing wave(s) can also be tuned to allow passage of cells if
desired, as well as allow the passage of fines/debris. This tuning
for material passage permits the acoustic standing wave(s) to
perform a cleaning operation for the bioreactor. Continuous,
steady-state operation is possible without pressure fluctuations,
and the product stream does not accumulate in the bioreactor or the
filtering device.
[0178] The acoustic perfusion device can be made of appropriate
materials known in the art. Such materials include high density
polyethylene (HDPE), other plastics, and potentially metals and
glasses. It has been found very convenient for the device to be
transparent, so that fluid flow and ultrasonic transducer operation
can be visually confirmed.
[0179] FIG. 5 shows another embodiment of an acoustic perfusion
device 500. This embodiment is very similar to the device 400
depicted in FIG. 4. The main difference is that the acoustic
perfusion device 500 of FIG. 5 has a first ultrasonic transducer
460 on one sidewall of the device and a second ultrasonic
transducer 562 on an opposite sidewall 440 thereof in the
collection zone 456. Put another way, the two transducers 460, 562
are located on opposite sides of the collection flow path 453. With
this arrangement, the reflector 580 is located within the
collection zone 456 between the first and second ultrasonic
transducers 460, 562. The transducers are oriented so that the
reflector 580 and first and second ultrasonic transducers 460, 562
create multi-dimensional standing wave(s) in the fluid cell 450 as
described above, or put another way the transducers are facing each
other. Also illustrated is the outflow pump 592 attached to the
outlet port 430 of the device, which is used to control the flow
rate of the fluid mixture flowing through the device. Not
illustrated here is the pump attached to the collection ports (not
visible) of the filtering device 500.
[0180] Turning now to FIG. 6, a processing system is shown
including an associated bioreactor 610 and an acoustic perfusion
device 630 of the present disclosure. The system is set up for use
as a perfusion bioreactor. The bioreactor 610 includes a reaction
vessel 620 having a feed inlet 622, an outlet 624, and a recycle
inlet 626. Fresh media is added into the feed inlet 622 by an
addition pipe 650. Some reactors will also include an outlet or
bleed port (not shown here) to remove or "bleed" cells in order to
maintain a constant cell density within a reactor. The contents of
the reaction vessel (reference numeral 605) are mixed with an
agitator 625. The desired product (e.g., recombinant proteins) is
continuously produced by cells located within the vessel 620, and
are present in the media of the bioreactor. The product and the
cells in the perfusion bioreactor are drawn from the reaction
vessel through pipe 652, and enter the acoustic perfusion device
630 through inlet port 632. Therein, a portion of the desired
product is separated from the cells. The desired product can be
drawn off through a first collection port 634 (which is a product
recovery port) and pipe 654 into a containment vessel 640, or in
the case of a truly continuous production system, some other
downstream purification process. The cells are returned to the
perfusion bioreactor after separation, passing from outlet port 636
(which is a fluid recycle port) of the acoustic perfusion device
through pipe 656 to recycle inlet 626 of the reaction vessel, which
form a recycle path. The multi-dimensional standing wave(s) of the
acoustic perfusion device are used to create a separation barrier
between the fluid cell of the device and the collection port, so
that a highly reduced number of biological cells are collected in
collection port 634.
[0181] FIG. 7 and FIG. 8 are views of another exemplary embodiment
of an acoustic perfusion device. FIG. 7 is a front cross-sectional
view, and FIG. 8 is an exterior perspective view. Notably, this
embodiment is specifically designed such that it can be fabricated
with clean machining techniques, using Class VI materials (medical
device grade HDPE, for example), or even as single or welded
injection molded part. In this manner, this embodiment is an
example of a single-use device, which is gamma-stable. The devices
are flushed to remove bioburden and then gamma-irradiated
(generally from 25-40 kGy) to sterilize any potential contamination
that could destroy a healthy cell culture, such as that present in
a perfusion bioreactor.
[0182] Referring first to FIG. 7, in this device 700, the inlet
port 710 and the collection port 770 are both located at the top
end 718 of the device, or on the top wall 776 of the device. The
outlet port 730 is located at a bottom end 716 of the device. Here,
the inlet port 710 and the outlet port 730 are both on a first side
712 of the device. The inlet flow path 751 is in the form of a
channel 755 that runs from the inlet port downwards towards the
bottom end and past the outlet port, the channel being separated
from the acoustic chamber 750 (here, the separation occurring by an
internal wall 756). Fluid will flow downwards in the channel, then
rise upwards into the acoustic chamber 750. The bottom wall 720 of
the acoustic chamber is a sloped planar surface that slopes down
towards the outlet port 730. The location of the ultrasonic
transducers 760 are shown here as two squares, between the top end
and the bottom end of the device. The collection flow path 753 is
located above the transducers.
[0183] Referring now to FIG. 8, the device 700 is shown as being
formed within a three-dimensional rectangular housing 706. It can
be seen that the outlet port 730 at the bottom end 716 of the
device is located on a front wall 775. Again, the collection port
770 and the inlet port 710 are located on a top wall 776. A viewing
window 708 made of a transparent material is present in the front
wall. Through that viewing window, it can be seen that the
ultrasonic transducers are mounted in the rear wall 778 of the
device housing. The viewing window acts as a reflector to generate
the multi-dimensional acoustic standing waves.
[0184] FIG. 9 and FIG. 10 are views of yet another exemplary
embodiment of an acoustic perfusion device. FIG. 9 is a front
cross-sectional view, and FIG. 10 is a perspective view.
[0185] Referring first to FIG. 9, in this device 900, there is an
inlet port 910 present on a front side 975 of the device along the
first side 912 of the device. An outlet port 930 (best seen in FIG.
10) is located directly opposite and at the same height as the
inlet port 910, and is also located on first side 912. In this
embodiment, there is a main fluid stream that flows almost directly
from the inlet port 910 to the outlet port 930, and the inlet flow
path 951 diverts a small side flow into the acoustic chamber 950
from the first side 912 of the device. The collection port 970 is
located at the top end 918 of the device, or on the top wall 976 of
the device. A secondary outlet port 980 is located on the first
side 912 of the device as well, extending from first side wall 979,
and located below the inlet port 910, and can act as a bleed port.
The bottom wall 920 of the acoustic chamber is shaped in a
pyramid-like fashion to taper downwards to a vertex. A drain line
981 runs from the bottom of the acoustic chamber 950 to the
secondary outlet port 980. It is contemplated that here, the
secondary outlet port can be used to capture a small flow of highly
concentrated cells, which can either be discarded (cell bleed) or
can also be returned back to the bioreactor.
[0186] Referring now to FIG. 10, the front wall 975 of the device
has a rectangular space 960, and the rear wall 978 of the device
has a rectangular space 962. It is contemplated that one transducer
and one reflector can be placed in these two rectangular spaces
960/962 in either orientation, or that two transducers could be
placed in the two rectangular spaces. The inlet port 910 and outlet
port 930 are both visible in this view. The inlet port 910 is
located on the front side of the device, and the outlet port 930 is
located on the rear side of the device (though this arrangement
could be reversed if desired). The clarification flow path 953 is
located above the transducers. Although not depicted here, a
mounting piece similar to that in FIG. 4 could be attached to the
second side 914 of the device.
[0187] FIGS. 43-45 are views of yet another exemplary embodiment of
an acoustic perfusion device. FIG. 43 is a perspective view, FIG.
10 is a picture showing a front view, and FIG. 45 is a side view.
FIG. 46A/B and FIG. 46C/D are schematic front views of possible
interior layouts of the device. FIG. 46A and FIG. 46B are
identical, and are used because of the large number of reference
numerals. FIG. 46C and FIG. 46D are also identical
[0188] Referring now to FIGS. 43-45, in this device 4300, the inlet
port 4310 and the outlet port 4330 are both located at the bottom
end 4316 of the device, and the collection port 4370 is located at
the top end 4318 of the device. The inlet port 4310 is located on a
first side 4312 of the device, and the outlet port 4330 is located
on a second side 4314 of the device. In FIG. 43, the outlet port
4330 is attached to a pump 4305, which creates flow through the
device 4300. A viewing window 4308 is present on the front wall
4375 of the device. The front wall 4375, top wall 4376, rear wall
4378, and first side wall 4379 are part of the housing 4306 that
surrounds the interior of the device.
[0189] Referring now to FIG. 43 and FIG. 45, the ultrasonic
transducer 4360 is located on the rear wall 4378 at the top end
4318 of the device. The viewing window 4308 acts as a reflector to
generate the multi-dimensional acoustic standing waves.
[0190] In this embodiment, a recirculation pipe 4340 connects the
inlet port 4310 directly to the outlet port 4330, and forms a
recirculation flow path (arrow 4356) through which cell culture
media containing cells and other materials can be continuously
recirculated through the perfusion device without entering the
acoustic chamber 4350. The recirculation pipe 4340 and the
recirculation flow path 4356 are located below the acoustic chamber
4350.
[0191] An inflow passageway 4380 and an outflow passageway 4390
connect the acoustic chamber 4350 to the recirculation pipe 4340,
and split off a portion of the flow of cell culture media from the
recirculation pipe into the acoustic chamber. Arrow 4351 indicates
the inlet flow path, and arrow 4355 indicates the outlet flow path.
These two passageways are particularly visible in FIG. 44. Put
another way, the inlet flow path travels through a different
passage than the outlet flow path. This arrangement creates a
secondary recirculating flow that is tangential to the acoustic
interface, and allows for constant recirculation of cells beneath
this acoustic interface, traveling in the same net direction as the
recirculation flow path 4356.
[0192] The flow geometry of the inflow passageway 4380 and the
outflow passageway 4390 can affect the flow profile through the
acoustic chamber. FIG. 46A and FIG. 46C are front views showing two
different internal structures that result in different flow
profiles. In these two figures, the inlet port 4310 is on the
right, and the outlet port 4330 is on the left.
[0193] Considering FIG. 46A and FIG. 46B first, the acoustic
chamber 4350 is shown, with the ultrasonic transducer 4360 shown in
dashed line. The acoustic chamber 4350 includes a first side wall
4362 and a second side wall 4364. The inflow passageway 4380 also
has a first wall 4381 and a second wall 4382, with the first wall
4381 extending beyond the first side wall 4362, or closer to the
inlet port 4310. The bottom cross-sectional area of the inflow
passageway (adjacent the recirculation pipe 4340) is indicated by
reference numeral 4384, and the top cross-sectional area of the
inflow passageway (adjacent the acoustic chamber 4350) is indicated
by reference numeral 4383. In embodiments, the top cross-sectional
area of the inflow passageway is greater than the bottom
cross-sectional area of the inflow passageway.
[0194] The outflow passageway 4390 also has a first wall 4391 and a
second wall 4392. The first wall 4391 and the second wall 4392
taper towards each other from the acoustic chamber 4350 to the
recirculation pipe 4340. The bottom cross-sectional area of the
outflow passageway (adjacent the recirculation pipe 4340) is
indicated by reference numeral 4394, and the top cross-sectional
area of the outflow passageway (adjacent the acoustic chamber 4350)
is indicated by reference numeral 4393. In embodiments, the top
cross-sectional area of the outflow passageway is greater than the
bottom cross-sectional area of the outflow passageway.
[0195] It is noted that the top cross-sectional area 4393 of the
outflow passageway is greater than the top cross-sectional area
4383 of the outflow passageway. The bottom cross-sectional area
4394 of the outflow passageway is also less than the bottom
cross-sectional area 4384 of the outflow passageway. Desirably,
this arrangement promotes the direction for cells and other larger
materials to enter the acoustic chamber 4350, and maximizes their
opportunity to exit the acoustic chamber in the same direction as
the main recirculation flow.
[0196] Now considering FIG. 46C and FIG. 46D, the first wall 4381
of the inflow passageway 4380 is essentially in-line with the first
side wall 4362. The second wall 4382 is vertical like the first
side wall, then widens at the top. The top cross-sectional area
4383 of the inflow passageway is greater than the bottom
cross-sectional area 4384 of the inflow passageway. The first wall
4391 of the outflow passageway 4390 tapers downwards, and then
becomes vertical. The second wall 4392 tapers inwards from the
second side wall 4364 to the recirculation pipe 4340. Again, the
top cross-sectional area 4393 of the outflow passageway is greater
than the bottom cross-sectional area 4394 of the outflow
passageway. In FIG. 46C, the top cross-sectional area 4393 of the
outflow passageway is still greater than the top cross-sectional
area 4383 of the outflow passageway. The bottom cross-sectional
area 4394 of the outflow passageway can be about equal to or less
than the bottom cross-sectional area 4384 of the outflow
passageway.
[0197] FIGS. 47-49 are views of yet another exemplary embodiment of
an acoustic perfusion device. FIG. 47 is a perspective view, FIG.
48 is a side view. FIG. 49 is a front schematic view of the
interior layout of the device.
[0198] Referring now to FIGS. 47-49, in this device 4700, the inlet
port 4710 and the outlet port 4730 are both located at the bottom
end 4716 of the device, and the collection port 4770 is located at
the top end 4718 of the device. The inlet port 4710 is located on
the front wall 4775 of the device, and the outlet port 4730 is
located on the rear wall 4778 of the device. In FIG. 47, the outlet
port 4730 is attached to a pump 4705, which creates flow through
the device 4700. A viewing window 4708 is present on the front wall
4775 of the device. The front wall 4775, top wall 4776, and rear
wall 4778 are part of the housing 4706 that surrounds the interior
of the device.
[0199] Referring now to FIG. 47 and FIG. 48, the ultrasonic
transducer 4760 is located on the rear wall 4778 at the top end
4718 of the device. The viewing window 4708 acts as a reflector to
generate the multi-dimensional acoustic standing waves.
[0200] Again, a recirculation pipe 4740 connects the inlet port
4710 directly to the outlet port 4730, and forms a recirculation
flow path (arrow 4756) through which cell culture media containing
cells and other materials can be continuously recirculated through
the perfusion device without entering the acoustic chamber 4750.
The recirculation pipe 4740 and the recirculation flow path 4756
are located below the acoustic chamber 4750.
[0201] This embodiment differs from that of FIG. 43 in that a
single passageway 4772 connects the acoustic chamber 4750 to the
recirculation pipe 4740, rather than the two separate passageways
(4380, 4390) of FIG. 43. Referring now to FIG. 49, arrow 4751
indicates the inlet flow path, and arrow 4755 indicates the outlet
flow path, both traveling through the single passageway. This flow
still results in a secondary recirculating flow that is tangential
to the acoustic interface, and allows for constant recirculation of
cells beneath this acoustic interface, traveling in the same net
direction as the recirculation flow path 4756.
[0202] It may be helpful now to describe the ultrasonic
transducer(s) used in the acoustic filtering device in more detail.
FIG. 11 is a cross-sectional diagram of a conventional ultrasonic
transducer. This transducer has a wear plate 50 at a bottom end,
epoxy layer 52, ceramic piezoelectric element 54 (made of, e.g.
Lead Zirconate Titanate (PZT)), an epoxy layer 56, and a backing
layer 58. On either side of the ceramic piezoelectric element,
there is an electrode: a positive electrode 61 and a negative
electrode 63. The epoxy layer 56 attaches backing layer 58 to the
piezoelectric element 54. The entire assembly is contained in a
housing 60 which may be made out of, for example, aluminum. The
housing is used as the ground electrode. An electrical adapter 62
provides connection for wires to pass through the housing and
connect to leads (not shown) which attach to the piezoelectric
element 54. Typically, backing layers are designed to add damping
and to create a broadband transducer with uniform displacement
across a wide range of frequency and are designed to suppress
excitation of particular vibrational eigen-modes of the
piezoelectric element. Wear plates are usually designed as
impedance transformers to better match the characteristic impedance
of the medium into which the transducer radiates.
[0203] FIG. 12 is a cross-sectional view of an ultrasonic
transducer 81 according to an example of the present disclosure,
which is used in the acoustic filtering device of the present
disclosure. Transducer 81 is shaped as a disc or a plate, and has
an aluminum housing 82. The aluminum housing has a top end and a
bottom end. The transducer housing may also be composed of
plastics, such as medical grade HDPE or other metals. The
piezoelectric element is a mass of perovskite ceramic, each
consisting of a small, tetravalent metal ion, usually titanium or
zirconium, in a lattice of larger, divalent metal ions, usually
lead or barium, and O.sup.2- ions. As an example, a PZT (lead
zirconate titanate) piezoelectric element 86 defines the bottom end
of the transducer, and is exposed from the exterior of the bottom
end of the housing. The piezoelectric element is supported on its
perimeter by a small elastic layer 98, e.g. epoxy, silicone or
similar material, located between the piezoelectric element and the
housing. Put another way, no wear plate or backing material is
present. However, in some embodiments, there is a layer of plastic
or other material separating the piezoelectric element from the
fluid in which the acoustic standing wave is being generated. The
piezoelectric element/crystal has an exterior surface (which is
exposed) and an interior surface as well. In particular
embodiments, the piezoelectric element/crystal is an irregular
polygon, and in further embodiments is an asymmetrical irregular
polygon.
[0204] Screws 88 attach an aluminum top plate 82a of the housing to
the body 82b of the housing via threads. The top plate includes a
connector 84 for powering the transducer. The top surface of the
PZT piezoelectric element 86 is connected to a positive electrode
90 and a negative electrode 92, which are separated by an
insulating material 94. The electrodes can be made from any
conductive material, such as silver or nickel. Electrical power is
provided to the PZT piezoelectric element 86 through the electrodes
on the piezoelectric element. Note that the piezoelectric element
86 has no backing layer or epoxy layer. Put another way, there is
an interior volume or an air gap 87 in the transducer between
aluminum top plate 82a and the piezoelectric element 86 (i.e. the
housing is empty). A minimal backing 58 (on the interior surface)
and/or wear plate 50 (on the exterior surface) may be provided in
some embodiments, as seen in FIG. 13.
[0205] The transducer design can affect performance of the system.
A typical transducer is a layered structure with the ceramic
piezoelectric element bonded to a backing layer and a wear plate.
Because the transducer is loaded with the high mechanical impedance
presented by the standing wave, the traditional design guidelines
for wear plates, e.g., half wavelength thickness for standing wave
applications or quarter wavelength thickness for radiation
applications, and manufacturing methods may not be appropriate.
Rather, in one embodiment of the present disclosure the
transducers, there is no wear plate or backing, allowing the
piezoelectric element to vibrate in one of its eigenmodes with a
high Q-factor, or in a combination of several eigenmodes. The
vibrating ceramic piezoelectric element/disk is directly exposed to
the fluid flowing through the fluid cell.
[0206] Removing the backing (e.g. making the piezoelectric element
air backed) also permits the ceramic piezoelectric element to
vibrate at higher order modes of vibration with little damping
(e.g. higher order modal displacement). In a transducer having a
piezoelectric element with a backing, the piezoelectric element
vibrates with a more uniform displacement, like a piston. Removing
the backing allows the piezoelectric element to vibrate in a
non-uniform displacement mode. The higher order the mode shape of
the piezoelectric element, the more nodal lines the piezoelectric
element has. The higher order modal displacement of the
piezoelectric element creates more trapping lines, although the
correlation of trapping line to node is not necessarily one to one,
and driving the piezoelectric element at a higher frequency will
not necessarily produce more trapping lines.
[0207] It is contemplated that, in some embodiments of the acoustic
filtering device of the present disclosure, the piezoelectric
element may have a backing that minimally affects the Q-factor of
the piezoelectric element (e.g. less than 5%). The backing may be
made of a substantially acoustically transparent material such as
balsa wood, foam, or cork which allows the piezoelectric element to
vibrate in a higher order mode shape and maintains a high Q-factor
while still providing some mechanical support for the piezoelectric
element. The backing layer may be a solid, or may be a lattice
having holes through the layer, such that the lattice follows the
nodes of the vibrating piezoelectric element in a particular higher
order vibration mode, providing support at node locations while
allowing the rest of the piezoelectric element to vibrate freely.
The goal of the lattice work or acoustically transparent material
is to provide support without lowering the Q-factor of the
piezoelectric element or interfering with the excitation of a
particular mode shape.
[0208] Placing the piezoelectric element in direct contact with the
fluid also contributes to the high Q-factor by avoiding the
dampening and energy absorption effects of the epoxy layer and the
wear plate. Other embodiments of the transducer(s) may have wear
plates or a wear surface to prevent the PZT, which contains lead,
from contacting the host fluid. This additional layer may be
desirable in, for example, biological applications such as
separating blood, biopharmaceutical perfusion, or fed-batch
filtration of mammalian cells. Such applications might use a wear
layer such as chrome, electrolytic nickel, or electroless nickel.
Chemical vapor deposition could also be used to apply a layer of
poly(p-xylylene) (e.g. Parylene) or other polymer. Organic and
biocompatible coatings such as silicone or polyurethane are also
usable as a wear surface. Thin films, such as a PEEK film, can also
be used as a cover of the transducer surface exposed to the fluid
with the advantage of being a biocompatible material. In one
embodiment, the PEEK film is adhered to the face of the
piezomaterial using pressure sensitive adhesive (PSA). Other films
can be used as well.
[0209] In some embodiments, for applications such as oil/water
emulsion splitting and others such as perfusion, the ultrasonic
transducer has a nominal 2 MHz resonance frequency. Each transducer
can consume about 28 W of power for droplet trapping at a flow rate
of 3 GPM. This power consumption translates to an energy cost of
0.25 kW hr/m.sup.3. This cost is an indication of the very low cost
of energy of this technology. Desirably, each transducer is powered
and controlled by its own amplifier. In other embodiments, the
ultrasonic transducer uses a square piezoelectric element, for
example with 1''.times.1'' dimensions. Alternatively, the
ultrasonic transducer can use a rectangular piezoelectric element,
for example with 1''.times.2.5'' dimensions. Power dissipation per
transducer was 10 W per 1''.times.1'' transducer cross-sectional
area and per inch of acoustic standing wave span in order to get
sufficient acoustic trapping forces. For a 4'' span of an
intermediate scale system, each 1''.times.1'' square transducer
consumes 40 W. The larger 1''.times.2.5'' rectangular transducer
uses 100 W in an intermediate scale system. The array of three
1''.times.1'' square transducers would consume a total of 120 W and
the array of two 1''.times.2.5'' transducers would consume about
200 W. Arrays of closely spaced transducers represent alternate
potential embodiments of the technology. Transducer size, shape,
number, and location can be varied as desired to generate desired
multi-dimensional acoustic standing wave patterns.
[0210] In some examples, the size, shape, and thickness of the
transducer can determine the transducer displacement at different
frequencies of excitation. Transducer displacement with different
frequencies may affect separation efficiency. Typically, the
transducer is operated at frequencies near the thickness resonance
frequency (half wavelength). Gradients in transducer displacement
typically result in more trapping locations for the
cells/biomolecules. Higher order modal displacements can generate
three-dimensional acoustic standing waves with strong gradients in
the acoustic field in all directions, thereby creating strong
acoustic radiation forces in all directions, which forces may, for
example, be equal in magnitude, leading to multiple trapping lines,
where the number of trapping lines correlate with the particular
mode shape of the transducer.
[0211] To investigate the effect of the transducer displacement
profile on acoustic trapping force and separation efficiencies, an
experiment was repeated ten times using a 1''.times.1'' square
transducer, with all conditions identical except for the excitation
frequency. Ten consecutive acoustic resonance frequencies,
indicated by circled numbers 1-9 and letter A on FIG. 14, were used
as excitation frequencies. The conditions were experiment duration
of 30 min, a 1000 ppm oil concentration of approximately 5-micron
SAE-30 oil droplets, a flow rate of 500 ml/min, and an applied
power of 20 W. Oil droplets were used because oil is less dense
than water, and can be separated from water using
acoustophoresis.
[0212] FIG. 14 shows the measured electrical impedance amplitude of
a square transducer as a function of frequency in the vicinity of
the 2.2 MHz transducer resonance. The minima in the transducer
electrical impedance correspond to acoustic resonances of the water
column and represent potential frequencies for operation.
Additional resonances exist at other frequencies where
multi-dimensional standing waves are excited. Numerical modeling
has indicated that the transducer displacement profile varies
significantly at these acoustic resonance frequencies, and thereby
directly affects the acoustic standing wave and resulting trapping
force. Since the transducer operates near its thickness resonance,
the displacements of the electrode surfaces are essentially out of
phase. The typical displacement of the transducer electrodes may
not be uniform and varies depending on frequency of excitation. As
an example, at one frequency of excitation with a single line of
trapped oil droplets, the displacement has a single maximum in the
middle of the electrode and minima near the transducer edges. At
another excitation frequency, the transducer profile has multiple
maxima leading to multiple trapped lines of oil droplets. Higher
order transducer displacement patterns result in higher trapping
forces and multiple stable trapping lines for the captured oil
droplets.
[0213] As the oil-water emulsion passed by the transducer, the
trapping lines of oil droplets were observed and characterized. The
characterization involved the observation and pattern of the number
of trapping lines across the fluid channel, as shown in FIG. 15,
for seven of the ten resonance frequencies identified in FIG. 14.
Different displacement profiles of the transducer can produce
different (more) trapping lines in the standing waves, with more
gradients in displacement profile generally creating higher
trapping forces and more trapping lines. Different displacement
profiles of the transducer can produce different (more) trapping
lines in the standing waves, with more gradients in displacement
profile generally creating higher trapping forces and more trapping
lines. It is noted that although the different trapping line
profiles shown in FIG. 15 were obtained at the frequencies shown in
FIG. 14, these trapping line profiles can also be obtained at
different frequencies. FIG. 15 shows the different crystal
vibration modes possible by driving the crystal to vibrate at
different fundamental frequencies of vibration. The 3D mode of
vibration of the crystal is carried by the acoustic standing wave
across the fluid in the chamber all the way to the reflector and
back. The resulting multi-dimensional standing wave can be thought
of as containing two components. The first component is a planar
out-of-plane motion component (uniform displacement across crystal
surface) of the crystal that generates a standing wave, and the
second component is a displacement amplitude variation with peaks
and valleys occurring in lateral directions across the crystal
surface. Three-dimensional force gradients are generated by the
standing wave. These three-dimensional force gradients result in
lateral radiation forces that stop and trap the particles with
respect to the flow by overcoming the viscous drag force. In
addition, the lateral radiation forces are responsible for creating
tightly packed clusters of particles. Therefore, particle
separation and gravity-driven collection depends on generating a
multi-dimensional standing wave that can overcome the particle drag
force as the mixture flows through the acoustic standing wave.
Multiple particle clusters are formed along trapping lines in the
axial direction of the standing wave, as presented schematically in
FIG. 15.
[0214] FIG. 16 is a numerical model showing a pressure field that
matches the 9 trapping line pattern. The numerical model is a
two-dimensional model; and therefore three trapping lines are
observed. Two more sets of three trapping lines exist in the third
dimension perpendicular to the plane of the page.
[0215] The lateral force of the acoustic radiation force generated
by the transducer can be increased by driving the transducer in
higher order mode shapes, as opposed to a form of vibration where
the crystal effectively moves as a piston having a uniform
displacement. The acoustic pressure is proportional to the driving
voltage of the transducer. The electrical power is proportional to
the square of the voltage. The transducer is typically a thin
piezoelectric plate, with electric field in the z-axis and primary
displacement in the z-axis. The transducer is typically coupled on
one side by air (i.e., the air gap within the transducer) and on
the other side by the fluid mixture of the cell culture media. The
types of waves generated in the plate are known as composite waves.
A subset of composite waves in the piezoelectric plate is similar
to leaky symmetric (also referred to as compressional or
extensional) Lamb waves. The piezoelectric nature of the plate
typically results in the excitation of symmetric Lamb waves. The
waves are leaky because they radiate into the water layer, which
result in the generation of the acoustic standing waves in the
water layer. Lamb waves exist in thin plates of infinite extent
with stress free conditions on its surfaces. Because the
transducers of this embodiment are finite in nature, the actual
modal displacements are more complicated.
[0216] FIG. 17 shows the typical variation of the in-plane
displacement (x-displacement) and out-of-plane displacement
(y-displacement) across the thickness of the plate, the in-plane
displacement being an even function across the thickness of the
plate and the out-of-plane displacement being an odd function.
Because of the finite size of the plate, the displacement
components vary across the width and length of the plate. In
general, a (m,n) mode is a displacement mode of the transducer in
which there are m undulations in transducer displacement in the
width direction and n undulations in the length direction, and with
the thickness variation as described in FIG. 17. The maximum number
of m and n is a function of the dimension of the crystal and the
frequency of excitation. Additional three-dimensional modes exist
that are not of the form (m,n).
[0217] The transducers are driven so that the piezoelectric element
vibrates in higher order modes of the general formula (m, n), where
m and n are independently 1 or greater. Generally, the transducers
will vibrate in higher order modes than (2,2). Higher order modes
will produce more nodes and antinodes, result in three-dimensional
standing waves in the water layer, characterized by strong
gradients in the acoustic field in all directions, not only in the
direction of the standing waves, but also in the lateral
directions. As a consequence, the acoustic gradients result in
stronger trapping forces in the lateral direction.
[0218] The transducer can be driven by a signal, such as a voltage
signal, a current signal, a magnetic signal, an electromagnetic
signal, a capacitive signal, or any other type of signal to which
the transducer is responsive to create a multi-dimensional acoustic
standing wave. In embodiments, the voltage signal driving the
transducer can have a sinusoidal, square, sawtooth, pulsed, or
triangle waveform; and have a frequency of 500 kHz to 10 MHz. The
voltage signal can be driven with pulse width modulation, which
produces any desired waveform. The voltage signal can also have
amplitude or frequency modulation start/stop capability to
eliminate streaming.
[0219] The transducers are used to create a pressure field that
generates acoustic radiation forces of the same order of magnitude
both orthogonal to the standing wave direction and in the standing
wave direction. When the forces are roughly the same order of
magnitude, particles of size 0.1 microns to 300 microns will be
moved more effectively towards "trapping lines", so that the
particles will not pass through the pressure field and continue to
exit through the collection ports of the filtering device. Instead,
the particles will remain within the acoustic chamber to be
recycled back to the bioreactor.
[0220] In biological applications, it is contemplated that all of
the parts of the system (i.e., the bioreactor, acoustic filtering
device, tubing fluidly connecting the same, etc.) can be separated
from each other and be disposable. Avoiding centrifuges and filters
allows better separation of the CHO cells without lowering the
viability of the cells. The transducers may also be driven to
create rapid pressure changes to prevent or clear blockages due to
agglomeration of CHO cells. The frequency of the transducers may
also be varied to obtain optimal effectiveness for a given
power.
[0221] The following examples are provided to illustrate the
devices, components, and methods of the present disclosure. The
examples are merely illustrative and are not intended to limit the
disclosure to the materials, conditions, or process parameters set
forth therein.
EXAMPLES
Example 1
[0222] FIG. 18 shows an experimental setup for an acoustic
perfusion device as described in detail above. This acoustic
perfusion device is very similar to that illustrated in FIG. 5,
except the bottom wall is not curved, but rather runs horizontally
from the first end and then angles directly to the outlet port.
Tubes are connected to the inlet port, outlet port, and the two
collection ports. A pump is also visibly fluidly connected to the
outlet port.
[0223] FIG. 19 is a picture of another acoustic perfusion device of
the present disclosure, similar to the embodiment shown in FIG. 5,
having two ultrasonic transducers and a concave bottom wall leading
from the inlet port to the outlet port at the bottom end of the
device. A cell containing fluid mixture is also present in the
device. In this picture, acoustic standing waves are created in the
collection zone between the reflector and first and second
transducers as described above. The acoustic field generated
thereby is indicated by waves and reference numeral 1664. The flow
pattern of the fluid mixture through the device from the inlet port
to the outlet port is shown with an arrow (reference numeral 1610)
indicating the direction of fluid flow into the device and arrows
(reference numeral 1630) indicating the direction of fluid flow
through the device towards the outlet port. Finally, the general
flow pattern of the desired product out of the device through the
first and second collection ports is shown with arrows (reference
numeral 1670) indicating the direction of flow.
[0224] Acoustophoretic separation has been tested using the
acoustic perfusion device of FIG. 19 and the methods of separation
of the present disclosure on different lines of Chinese hamster
ovary (CHO) cells. FIGS. 20-28 show various test results varying
different parameters and measuring different values using a Beckman
Coulter Cell Viability Analyzer.
[0225] The perfusion flow rates with the acoustic filtering device
were from about 2 mL/min to about 10 mL/min, or the flow rates were
about 1 VVD to about 5 VVD for a 2.7 L working volume bioreactor.
The VVD refers to the "vessel volume per day", or how many times
the volume of the bioreactor vessel is cycled through the acoustic
filtering device in one day. The perfusion flow rate (Qp) was
collected through the perfusion ports. In contrast, the feed flow
rates (Qf) were from about 40 mL/min to about 200 mL/min.
[0226] The feed solution had a starting CHO cell density of
50.times.10.sup.6 cells/mL. The reactor size was 2.7 L and the feed
volume of the host fluid was 1.5 L. In total, a series of seven
tests (T1-T7) were performed to study the effect of varying the VVD
and flow split in a 2.7 L volume reactor. The parameters for the
tests are shown in Table 1 below.
TABLE-US-00001 TABLE 1 System results for a 2.7 L reactor and feed
volume of 1.5 L Flow Split Qp Qf (Qp/Qf) 1 VVD 1.5 VVD 2 VVD 5.2
VVD 5.0% T1 T2 T3 T7 2.5% T4 T5 T6
[0227] The results included a cell clarification efficiency between
89-93% at a DC voltage of 45V, regardless of the flow rate as shown
in FIG. 20. It is noted that the DC voltage for T1 was fixed at
60V, whereas for tests T2-T7 the DC voltage was reduced to a fixed
amount of 45V. The transducer voltage amplitude is estimated to be
about half of these values.
[0228] The results further included a perfusate turbidity reduction
of 90-94% compared to the feed, as shown in FIG. 21. This figure
shows the turbidity of the feed, the recirculated fluid (Qc), and
the perfusate (Qp). The feed entered the inlet port, the
recirculated fluid exited the outlet port and was recirculated, and
the perfusate exited the perfusion port of the acoustic filtering
device. It is noted that the turbidity measurements for tests T1
and T2 resulted in a hardware error, and their results are not
displayed. The results of tests T3-T7 are displayed, which equated
to a 6-10% turbidity in the perfusion stream relative to the feed
stream, regardless of flow rate.
[0229] FIG. 22 was produced by a Beckman Coulter Cell Viability
Analyzer and revealed a cell viability for each flow rate that was
within the error of instrument (i.e., .+-.6%), with the control
ranging from 79-84% over all tests.
[0230] Further testing was performed using a solution designated
"CHO Line A". The solution had a starting cell density of
50.times.10.sup.6 cells/mL, a turbidity of 2,400 NTU, and cell
viability of roughly 80%. The solution was separated using a device
of the present disclosure in a system having a reactor size of 2.7
L. The volume of the feed fluid was between 1.5 L and 2.0 L. The
perfused flow rates were from 2 mL/min to 10 mL/min, or from 1 to 5
VVD. A series of six tests were performed to study the effect of
varying the VVD and flow split on acoustic filtration performance
for the 2.7 L volume reactor. The parameters for the tests are
shown in Table 2 below.
TABLE-US-00002 TABLE 2 System results for a 2.7 L reactor and feed
volume from 1.5 L-2.0 L T1 T2 T3 VVD 1.5 VVD 2 VVD 1 Flow Split
5.00% Flow Split 5.00% Flow Split 2.50% Perfused Flow 2.8 Perfused
Flow 3.8 Perfused Flow 1.9 (ml/min) (ml/min) (ml/min) Feed Flow
(ml/min) 56 Feed Flow (ml/min) 75 Feed Flow (ml/min) 75 T4 T5 T6
VVD 1.5 VVD 2 VVD 5.2 Flow Split 2.50% Flow Split 2.50% Flow Split
5.00% Perfused Flow 2.8 Perfused Flow 3.8 Perfused Flow 10 (ml/min)
(ml/min) (ml/min) Feed Flow (ml/min) 112.5 Feed Flow (ml/min) 150
Feed Flow (ml/min) 194.2
[0231] FIG. 23 shows the measured total cell density of feed flow,
recirculation flow, and perfused flow. The bioreactor cell
retention for the tests shows an approximately 90% perfusion
efficiency. FIG. 24 shows the measured cell viability for the
tests, revealing no significant change in viability across the
tests.
[0232] Next, additional testing was performed using a solution
designated "CHO Line B". The solution had a starting cell density
of 75.times.10.sup.6 cells/mL, a turbidity of 2,300 NTU, and cell
viability of roughly 80%. The solution was separated using a device
of the present disclosure in a system having a reactor size of 2.7
L. Four tests were performed (T1-T4). Two of the tests (T1, T3)
used a device having a single transducer. The other two tests (T2,
T4) used a device having two transducers in series (such that the
fluid ran through both standing waves). The parameters for the
tests are shown in Table 3 below.
TABLE-US-00003 TABLE 3 System results for a 2.7 L reactor and feed
volume from 1.5 L-2.0 L T1 T2 Transducers 1 Transducers 2 VVD 1 VVD
1 Perfused Flow (mL/min) 1.9 Perfused Flow (mL/min) 1.9 Feed Flow
(mL/min) 75 Feed Flow (mL/min) 75 T3 T4 Transducers 1 Transducers 2
VVD 2 VVD 2 Perfused Flow (mL/min) 3.8 Perfused Flow (mL/min) 3.8
Feed Flow (mL/min) 150 Feed Flow (mL/min) 150
[0233] FIG. 25 shows the measured total cell density of feed,
recirculation, and perfusion flows. The bioreactor cell retention
for the tests shows a perfusion efficiency greater than 90%. The
results further evidenced an approximately 3-5% greater efficiency
when using two transducers rather than a single transducer. FIG. 26
shows the measured cell viability for the tests, revealing no
significant change in viability across the tests. Practically
speaking, operating at low VVD offers a number of advantages, such
as media cost reduction.
Example 2
[0234] FIG. 27 shows another experimental setup for an acoustic
perfusion device similar to that illustrated in FIG. 8. Tubes are
connected to the inlet port, outlet port, and the collection
port.
[0235] The device was tested at a transducer voltage of 40V peak to
peak, a perfused flow rate of 15-30 mL/min, and a recirculation
flow rate of 2 L/min. Samples were taken every 45-60 minutes, and
the cell retention rate was determined. FIG. 28 shows the results.
The y-axis is the retention, stated in terms of percentage
(calculated by comparing the output cell count with the input, or
bioreactor/culture cell count). The x-axis is both the applied DC
voltage (in V) and the perfused, or harvest, flow rate (in mL/min);
it is merely coincidental that the range of numeric values for V
and mL/min are the same. The cell retention efficiency remained
above 95% for perfused flow rates up to 20 mL/min, and remained
above 90% up to about 25 mL/min. FIG. 33 is a composite picture
showing the device in a startup or cell settling mode (left) and in
a steady state cell retention mode (right).
[0236] Next, experiments were performed to determine what factors
would affect cell retention. The perfused flow rate was varied, as
was the transducer voltage. When the perfused flow rate was varied,
the transducer voltage was maintained at 40V peak to peak and the
recirculation flow rate was maintained at 2 L/min. When the
transducer voltage was varied, the perfused flow rate was
maintained at 20 mL/min and the recirculation flow rate was
maintained at 2 L/min. The results indicated that, for this
particular embodiment, a perfused flow rate of about 15 mL/min to
about 28 mL/min was optimum, and a transducer voltage of about 15V
peak to peak to about 28V peak to peak was optimum.
[0237] A better understanding of the added functionality provided
by an acoustic perfusion device can be demonstrated by examining
the observed cell samples coming in and being harvested from the
device. FIG. 29A is a microscope image (from a Vi-Cell cell
counter) of the feed suspension, here a viable cell culture
population with approximately 56 million cells/mL. Several large,
round, healthy cells can be observed. FIG. 29B is a histogram
showing the distribution of cell diameter in the population. The
diameter distribution is strongly bi-modal, around values of about
11 microns and about 23 microns. These two modes correspond roughly
with the smaller debris and non-viable cells, and the larger viable
cells. It should be noted that this sample is from a particularly
"dirty" cell population. In general, a production cell line would
be far cleaner, and the peak at approximately 11 microns would be
much smaller, or even non-existent.
[0238] FIG. 30A is another microscope image (from a Vi-Cell cell
counter), this time of the flow harvested from the acoustic
perfusion device. In this image, very few bright, large cells are
observed, in contrast to FIG. 29A. Rather, the image is filled with
more smaller, darker particles, or debris. The experimental
conditions in this case were a perfused rate of 4 mL/min and a
recirculation rate of 2 L/min with a DC input voltage of 30V. This
qualitative observation is confirmed by the histogram in FIG. 30B,
which shows the distribution of diameters in the perfusate. Looking
at FIG. 30B, the particle distribution is now unimodal, with the
peak of approximately 9 microns. This distribution indicates that
the larger, viable cells have been trapped and retained, or
otherwise largely prevented from exiting in the perfusate. Smaller
cells are passing through, together with cell debris, fines, and
fragments, from submicron to micron sized.
[0239] A computational fluid dynamics (CFD) model was made of this
device. FIG. 31 shows the velocity distributions within the device
after 500 seconds. The units are in meters/second (m/s). As
expected, the highest velocities are found in the channel leading
downwards from the inlet port to the outlet port. The velocity is
near zero in the fluid cell and out through the collection port.
This velocity profile is important for two reasons: the acoustic
field is more effective in a flow with a lower, more uniform
velocity, and because the cells used in bio-manufacturing are
sensitive to flow, and the induced shear rate.
[0240] FIG. 32 is a diagram illustrating several aspects of this
embodiment. Fluid flows into the device through the inlet port 710
(arrow 780) and into the acoustic chamber 790. The volume of fluid
750 below the acoustic chamber contains the tangential flow path,
indicated by arrow 782. Fluid with a relatively high amount of
viable cells will exit through the outlet port 730, as indicated by
arrow 781. The acoustic interface effect/region created by the
standing waves is marked with reference numeral 783, and is
upstream of the acoustic standing wave field 784. The acoustic
interface roughly coincides with an x-y plane in this diagram. This
interface effect separates large cells from smaller cell fragments,
particulate debris, desired biomolecules, etc., which can pass
through the interface 783 and the acoustic standing wave field 784.
By way of comparison, the cell aggregates that arise within the
acoustic standing wave field 784 during the first mode of operation
(see FIG. 41) can be described as being aligned in the y-z plane.
In operation, the separation caused by the interface effect occurs
at the interface region 783 as any large cells are held back by the
"interface" or "barrier" effect. The harvest flow stream 785
containing the smaller fragments, particulate debris, desired
biomolecules, etc. then exits through harvest port 770. The
tangential flow path is part of the inlet flow path, and is located
below the interface region 783 generated by the acoustic standing
wave. The tangential flow path will transport away both the
clusters of cells that drop from the acoustic standing wave field
784 due to gravity effects and the cells that are retained by the
acoustic interface effect.
[0241] FIG. 61 shows a device 6100 that is substantially similar to
the device 700 of FIG. 7 and FIG. 32. The same numbering is used in
FIG. 61. The main difference between the device illustrated in FIG.
32 and that illustrated in FIG. 61 is the flow of particles near
transducers 760, which is illustrated with arrow 6153, which runs
down past the transducers 760 and down to acoustic interface
effect/region 783. This flow pattern near transducers 760 occurs
because the particles in FIG. 61 are too small or have an acoustic
contrast factor that is insufficient to be affected by the acoustic
standing wave created by transducers 760 (and the reflector), and
so they can reenter the acoustic standing wave. This behavior may
also be affected by the buoyancy characteristics of the particles
and the fluid flow of the system, and thus the Stokes drag
experienced by the particles.
Example 3
[0242] Another way of explaining the operation of the acoustic
perfusion device can be understood by looking at the results of a
numerical study. In the numerical study, two fluids with differing
effective acoustic properties (i.e., speed of sound and density),
were modeled with an interface between the min COMSOL, a numerical
simulation software. The acoustic field is calculated and therefrom
the lateral radiation force acting on a particle in the direction
of the fluid velocity is calculated using Gorkov's equation.
[0243] FIG. 34 shows the geometry of the simulation, utilizing a
piezoelectric transducer, steel reflector, aluminum housing, and
two fluids: the first fluid being water within the acoustic field,
simulating the clarified fluid, and the second fluid being a 15%
concentration of CHO cells in water solution outside of the
acoustic field, the second fluid having a higher density and higher
speed of sound than the water fluid and simulating the bioreactor
fluid containing the cells.
[0244] The two fluids were separated as indicated by the solid line
in the model of FIG. 34. In this setup, the fluid velocity through
the system was in a horizontal direction from left to right.
Therefore, in order to act as a retention device, the acoustic
field needs to generate a force on the cells that acts in the
negative x-direction (i.e, opposite the fluid velocity). Water was
modeled with a fluid density of 1000 kg/m.sup.3 and a speed of
sound of 1500 m/s. CHO cells were modeled having a density of 1050
kg/m.sup.3 and a speed of sound of 1550 m/s. A coupled
multi-physics numerical simulation that included a full
piezoelectric simulation of the piezoelectric material, an acoustic
simulation of the two fluids, and a linear elastic simulation in
the steel and aluminum bodies was performed at various frequencies
of excitation. The transducer was driven at a peak voltage of 40
V.
[0245] FIGS. 35A-35C show the acoustic pressure in the two fluids
and the displacement of the piezoelectric material, the aluminum
housing, and the steel reflector of the model at frequencies of
operation of 2.218 MHz, 2.2465 MHz, and 2.3055 MHz. The lateral
radiation force (i.e., horizontally in the direction of the fluid
flow), was calculated at the interface between the two fluids along
with real electrical power consumed by the transducer. The
structural displacement of the transducer and steel are shown,
along with the acoustic pressure in the fluid.
[0246] FIG. 36 shows the lateral radiation force (N) and the
lateral radiation force normalized by power (N/W) versus frequency
acting on the suspended CHO cells. This graph shows that at the
resonance frequencies (i.e., local maxima in power), the average
lateral radiation force on the interface is negative, meaning that
it is in the negative-x direction. The result is the creation of an
acoustic barrier effect or an acoustic interface effect. That is,
the acoustic field at the interface between the two fluids exerts a
strong lateral force on the suspended particles in a direction
opposite to the fluid flow, thereby keeping the larger particles
from entering the acoustic field and allowing the first fluid
(i.e., fluid containing smaller particles, such as the desired
product, and excluding whole cells) to enter the acoustic field,
thereby creating an acoustic perfusion cell retention device. In
this way, the clarified fluid can escape and the cells are held
back by the radiation force. This force is never positive, meaning
that it always holds the cells back at the interface, not allowing
them to cross the interface. The multiple peaks in the power curve
show the existence of multiple modes of operation including planar
resonance modes and multi-dimensional modes of operation,
indicating that this type of operation can be generated through
utilization of planar and multi-dimensional standing waves alike.
In systems having 1''.times.1'' dimensions, there exists a planar
resonance about every 30 kHz. The graph shows evidence of
additional peaks indicating the existence of the multi-dimensional
modes. Per unit power, these modes can be equally or even more
effective as the planar resonance modes. As explained above, the
cells that are held back by the acoustic radiation force may then
be picked up by the scrubbing motion of the flow field (i.e., the
recirculating flow underneath the interface), and be continuously
returned to the bioreactor to ensure they receive the nutrition and
oxygen to maintain the production of the overall cell culture.
Example 4
[0247] FIG. 37 and FIG. 38 show another experimental setup for an
acoustic perfusion device similar to that illustrated in FIG. 9.
Tubes are connected to the inlet port, outlet port, the collection
port, and the secondary outlet port (for a flow concentrated
cells). Arrows are included to illustrate fluid flow. Arrows
indicate the flow into the inlet port; the flow out of the outlet
port; the perfusate flow out the top of the device and the flow of
concentrate out the bottom of the device. The flow through the
inlet port to the outlet port is the recirculation flowrate. The
perfusate flow out the top of the device is the perfused flowrate
containing clarified fluid depleted in cells and containing desired
product. The flow of concentrate out the bottom of the device is
the concentrated cell flow. The concentrated cell flow can be used
for a cell bleed operation or if desired, the cells can be returned
to the bioreactor.
[0248] The device was tested at a transducer voltage of 40V peak to
peak, a perfused flow rate (out the top) of 1-10 mL/min, a
recirculation flow rate of 0.75-1 L/min, and a concentrate flow
rate (out the bottom) of 15 mL/min. The cell retention rate was
determined for different perfused flowrates. FIG. 39 shows the
results. The y-axis is the retention with 1.00 indicating 100%
retention. The cell retention efficiency remained above 98% for
perfused flow rates up to 7 mL/min, and was just below 90% at 10
mL/min.
Example 5
[0249] The device of FIG. 7 and FIG. 8 was tested at different
flowrates on two different days. FIG. 50 shows the cell retention
(%) versus time. Here, higher values are more desirable, and most
values were over 95% at flowrates ranging from 1.5 mL/min to 8
mL/min. FIG. 51 shows the perfusate cell density (million cells/mL)
versus time. Here, lower values are more desirable (indicating
successful cell separation). As expected, results were better at
lower flowrates.
Example 6
[0250] A computational fluid dynamics (CFD) model was made of the
device with the internal structure of FIG. 46A. FIG. 52 shows the
velocity distributions within the device after 500 seconds. The
units are in meters/second (m/s). It is noted that here, the inlet
is from the left-hand side of the figure, and the outlet is on the
right-hand side of the figure (flow direction indicated by the
arrow). As expected, the highest velocities are found in the inflow
passageway into the acoustic chamber. Negative velocities in the
outflow passageway indicate flow out of the acoustic chamber. The
velocity is near zero in the acoustic chamber, and near the
collection port at the top thereof. This velocity profile is
desirable for perfusion operation.
[0251] A computational fluid dynamics (CFD) model was also made of
the device with the internal structure of FIG. 46C. FIG. 53 shows
the velocity distributions within the device at a flow rate of 250
mL/min, while FIG. 54 shows the velocity distributions within the
device at a flow rate of 1000 mL/min. In these two figures, the
inlet is from the right-hand side of the figure, and the outlet is
on the left-hand side of the figure (flow direction indicated by
the arrow). Again, the velocity is near zero in the acoustic
chamber, and near the collection port at the top thereof, even at
the much higher flow rate of 1000 mL/min. This velocity profile is
desirable for perfusion operation.
[0252] Two further CFD models were made of variants of the
configurations seen in FIG. 53 and FIG. 54, which are potential
internal structures for the device of FIG. 43. The first variant is
seen in FIG. 55. Here, the rectangle at the top indicates the
location of the ultrasonic transducer/multi-dimensional acoustic
standing wave. Below this rectangle, the sides of the acoustic
chamber taper evenly down to the outflow passageway. The inflow
passageway has an arcuate top into the acoustic chamber. The second
variant is seen in FIG. 56. This variant is much taller and
narrower compared to FIG. 55. The outflow side of the acoustic
chamber tapers evenly down to the outflow passageway. As seen in
both figures, the desirable velocity profile is present, being near
zero in the area of the multi-dimensional acoustic standing wave,
and near the collection port at the top thereof. These flow paths
of FIGS. 52-56 demonstrate how the fluid may be managed through
different configurations of the geometry leading from the main
recirculation path (through the recirculation pipe) to the acoustic
chamber. In FIG. 55 and FIG. 56, the main recirculation path to the
fluid chamber have the same flow rate. One consequence of these
configurations is that the separation velocities will be
higher.
[0253] Next, the device with the internal structure of FIG. 46A was
tested at different flowrates. FIG. 57 shows the cell retention (%)
versus time. Higher values are more desirable, and most values were
over 97% at flowrates ranging from 0.37 mL/min to 1.5 mL/min. FIG.
58 shows the perfusate cell density (million cells/mL) versus time.
Here, lower values are more desirable (indicating successful cell
separation). As expected, results were better at lower
flowrates.
[0254] The device was then tested using two different operating
frequencies for the ultrasonic transducer, 1 MHz or 2 MHz, and at
different flowrates. FIG. 59 shows the cell retention (%) versus
time. Higher values are more desirable. At 2 MHz, the values were
close to 100% for flow rates of 1.5 mL/min to 3 mL/min. At 1 MHz,
the values stayed over 90% for flow rates of 1.5 mL/min to 3
mL/min. The frequency of 2 MHz generally performed better. FIG. 60
shows the perfusate cell density (million cells/mL) versus time.
Again, results were better for 2 MHz operating frequency.
Example 7
[0255] Next, a device with an internal structure similar to FIG. 10
was used to perform acoustic separation of beads and yeast from a
host fluid. The device was a 1 inch by 1 inch by 1 inch device
using a 1 inch by 1 inch crystal to separate beads from a yeast
mixture having a concentration of 1.times.10.sup.6 cells/mL. The
ultrasonic transducer was operated at 1 MHz with an input voltage
of 60 volts, and the beads were Solohill microcarriers in the
amount of 7.5 g/L. The mixture was flowed into the device at a flow
rate of 75 mL/minute, while the concentrate was recovered at a rate
of 5 mL/minute. FIG. 62 shows a photograph of the microcarriers
being trapped in the acoustic standing wave within the device.
Example 8
[0256] Next, a device with an internal structure similar to FIG. 7
was used to examine AWS cell separation efficiency. FIG. 64 shows
the viable cell density (left-side y-axis), AWS power (right-side
y-axis), and harvest to recirculation ratio (right-side y-axis)
over a period of about 28 days. The dark dots represent the
bioreactor, while the light dots represent the AWS harvest. The
system was operated with AWS beginning on day 7 through the end of
the study. From day 7 to day 20, the system was operated at 30% AWS
power, before being switched to 10% AWS power from day 20 to the
end of the study. The harvest to recirculation ratio was about 15%
from day 7 (start of AWS) to day 11, about 30% from day 11 to the
end of the study.
[0257] The cell separation efficiency was dominated by the harvest
to recirculation flow ratio, as shown in FIG. 65 which compares the
AWS power (x-axis) to the harvest to recirculation flow ratio
(y-axis). The device was then paired with a 10 L bioreactor, and
the AWS cell separation efficiency was again examined. FIGS.
66A-66C show similar volumetric productivity, improved product
transmission, and improved product residence time for AWS versus
TFF. FIG. 67 shows the cell separation efficiency (right-side
y-axis), the harvest VCD (left-side y-axis), and bioreactor VCD
(left-side y-axis) over a period of about 14 days. The system had a
greater than 90% cell separation at 30% AWS power and a 1.25%
harvest to recirculation ratio.
[0258] Overall, AWS separation was shown to not affect the quality
between the bioreactor and the harvest, as shown in FIGS. 68A-68C.
FIG. 68A graphically shows the relative abundance after size
exclusion chromatography (SEC) of high molecular weight (HMW)
species, monomer species, and low molecular weight (LMW) species of
a monoclonal antibody (mAb). The darker bars represent the
bioreactor, and the lighter bars represent the harvest. FIG. 68B
graphically shows the relative abundance after ion exchange
chromatography (IEX) of acidic, main peak, and basic species of a
mAb. The darker bars represent the bioreactor, and the lighter bars
represent the harvest. FIG. 68C shows glycosylation of the protein.
The effects are the same for the protein that is from the
bioreactor and for the protein that has passed through the acoustic
separation device. These results show that acoustics do not affect
the protein.
[0259] Finally, AWS separation was shown to have comparable quality
to a TFF perfusion process using a 0.22 micron membrane, as shown
in FIG. 69A and FIG. 69B. FIG. 69A graphically shows the relative
abundance after size exclusion chromatography (SEC) of HMW species,
monomer species, and LMW species of a monoclonal antibody (mAb).
The darker bars represent AWS, and the lighter bars tangential flow
filtration (TFF). FIG. 69B graphically shows the relative abundance
after ion exchange chromatography (IEX) of acidic, main peak, and
basic species of a mAb. The darker bars represent AWS, and the
lighter bars represent TFF. As can be seen in FIG. 69A and FIG.
69B, there was a negligibly higher HWM species in AWS and similar
charge variance profile between AWS and TFF.
Example 9
[0260] FIG. 70 depicts another device 7000 that can be used for
acoustophoresis. Device 7000 includes a first inlet port 7002 along
a front wall 7011 of the device near the top end 7001 of the
device. A second inlet port (not shown) can be located along a rear
wall 7013 of the device opposite the front wall 7011 thereof. A
viewing window 7050 made of a transparent material is also present
in the front wall 7011. Through the viewing window 7050, it can be
seen that an ultrasonic transducer 7020 is also coupled to the rear
wall 7013. In this embodiment, the viewing window 7050 acts as a
reflector to generate the multi-dimensional acoustic standing
wave(s). Both the reflector and the ultrasonic transducer are
generally located below the inlet(s), such that the acoustic
chamber is positioned below the inlet(s). In particular
embodiments, the first and second inlets are located directly
opposite one another in the device. A drain outlet 7006 is located
near a bottom end 7003 of the device above the acoustic chamber,
and here is located along a first sidewall 7015 of the device. The
drain outlet generally serves as the outlet through which the
concentrate fluid (retained material prevented from passing through
the standing wave) is recovered from the device. A permeate outlet
7007, which generally serves as the outlet through which the
permeate (material that passes through the standing wave) is
recovered from the device, is located above the acoustic chamber,
and as illustrated here is also located along the first sidewall
7015 of the device above the drain outlet 7006.
[0261] The device 7000 includes a cooling unit 7060 coupled to the
rear wall 7013. The cooling unit 7060 includes an independent flow
path that is separate from the flow path through the device
containing the fluid that is to be exposed to the multi-dimensional
acoustic standing wave. A coolant inlet 7062 is adapted to permit
the ingress of a cooling fluid into the cooling unit. A coolant
outlet 7064 serves as the outlet through which the coolant and
waste heat exit the cooling unit. Here, the coolant inlet is
located below the coolant outlet, though this path can be varied as
desired. The coolant that flows through the cooling unit can be any
appropriate fluid. For example, the coolant can be water, air,
alcohol, ethanol, ammonia, or some combination thereof. The coolant
can, in certain embodiments, be a liquid, gas, or gel. The coolant
can be an electrically non-conductive fluid to prevent electric
short-circuits. The cooling unit can be used to cool the ultrasonic
transducer, which can be particularly advantageously when the
device is to be run continuously with repeated processing and
recirculation for an extended period of time (e.g., perfusion). The
cooling unit can also be used to cool the host fluid running
through the device 7000, if desired.
[0262] The performance of the device 7000 of FIG. 70 was tested
using Jurkat T cells and compared to a device having a structure
similar to device 900 of FIG. 9. The performance of each device is
summarized in the table below.
TABLE-US-00004 Feed (10.sup.6 Trapped Collected Volume Cell Device
cells/mL) (%) (%) Reduction Concentrating FIG. 9 0.90 65 49 5.7 2.8
FIG. 9 0.76 84 74 2.6 1.9 FIG. 70 0.77 86 71 11.9 8.4 FIG. 70 0.70
75 72 11.3 8.2 FIG. 70 0.77 87 77 11.4 8.9
[0263] The performance of the device was then tested using CHO
cells and again compared to a device having a structure similar to
device 900 of FIG. 9. The performance of each device is summarized
in the table below.
TABLE-US-00005 Feed (10.sup.6 Trapped Collected Volume Cell Device
cells/mL) (%) (%) Reduction Concentrating FIG. 70 1.11 91 72 12.8
9.2 FIG. 70 1.07 90 70 12.5 8.8 FIG. 70 0.99 92 67 13.0 8.7
[0264] The present disclosure has been described with reference to
exemplary embodiments. Modifications and alterations may occur to
others upon reading and understanding the preceding detailed
description. It is intended that the present disclosure be
construed as including all such modifications and alterations
insofar as they come within the scope of the appended claims or the
equivalents thereof.
* * * * *