U.S. patent application number 11/064252 was filed with the patent office on 2005-10-27 for stirred-tank reactor system.
This patent application is currently assigned to Baxter International, Inc.. Invention is credited to Kunas, Kurt T., Oakley, Robert V..
Application Number | 20050239198 11/064252 |
Document ID | / |
Family ID | 35242132 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239198 |
Kind Code |
A1 |
Kunas, Kurt T. ; et
al. |
October 27, 2005 |
Stirred-tank reactor system
Abstract
The present invention relates to a stirred-tank reactor system
and methods of preparing such systems. The present invention
further encompasses the use of the stirred-tank reactor system as a
disposable bioreactor and in kits with disposable elements.
Inventors: |
Kunas, Kurt T.; (Pleasanton,
CA) ; Oakley, Robert V.; (Lafayette, CA) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY
MAIL STOP DF2-2E
DEERFIELD
IL
60015
US
|
Assignee: |
Baxter International, Inc.
Deerfield
IL
Baxter Healthcare S.A.
Zurich
|
Family ID: |
35242132 |
Appl. No.: |
11/064252 |
Filed: |
February 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60565908 |
Apr 27, 2004 |
|
|
|
Current U.S.
Class: |
435/297.1 |
Current CPC
Class: |
B01F 15/00831 20130101;
C12M 27/02 20130101; C12M 23/06 20130101; C12M 23/28 20130101; B01F
2215/0073 20130101; C12M 23/26 20130101; C12M 23/00 20130101; Y10T
29/49826 20150115; B01F 7/22 20130101; B01F 7/1695 20130101; B01F
15/00006 20130101; C12M 23/14 20130101; C12M 29/06 20130101; B01F
15/0085 20130101; B01F 7/001 20130101 |
Class at
Publication: |
435/297.1 |
International
Class: |
C12M 001/12 |
Claims
What is claimed is:
1. A stirred-tank reactor system, comprising: (i) a flexible bag
with at least one opening, wherein the bag functions as a sterile
container for a fluidic medium; (ii) a shaft situated within the
bag; (iii) an impeller attachable to said shaft, wherein said
impeller is used to agitate the fluidic medium to provide a
hydrodynamic environment; and (iv) a bearing attached to said shaft
and to said opening of the bag.
2. The stirred-tank system of claim 1, wherein said system is
disposable.
3. The stirred-tank system of claim 1, wherein said system is
pre-sterilized.
4. The stirred-tank reactor system of claim 1, wherein said system
further comprises a housing on the outside of the bag, wherein said
housing includes at least one support that holds the bearing and a
motor, and wherein said bag is contained within the housing.
5. The stirred-tank reactor system of claim 4, wherein said system
further includes a heater selected from the group consisting of a
heating pad, a steam jacket, and a circulating fluid or water
heater.
6. The stirred-tank reactor system of claim 5, wherein said heater
is located between the bag and the housing.
7. The stirred-tank reactor system of claim 5, wherein said heater
is incorporated into the housing.
8. The stirred-tank reactor system of claim 4, wherein the housing
further includes a plurality of baffles and the bag folds around
the baffles.
9. The stirred-tank reactor system of claim 4, wherein said system
further includes a product loop with flow past a pH sensor and a
dissolved-oxygen sensor, wherein said sensors are incorporated into
the housing.
10. The stirred-tank reactor system of claim 4, wherein said
housing is selected from the group consisting of a metal barrel, a
plastic barrel, wood barrel, and a glass barrel.
11. The stirred-tank reactor system of claim 1, wherein said bag is
a disposable, plastic bag.
12. The stirred-tank reactor system of claim 1, wherein said
fluidic medium is selected from the group consisting of a
biological fluid, a cell culture medium, a culture of
microorganisms, and a food production.
13. The stirred-tank reactor system of claim 12, wherein the
fluidic medium is a cell culture medium and the system is operated
in batch mode, semi-batch mode, fed-batch mode, or continuous
mode.
14. The stirred-tank reactor system of claim 1, wherein said
impeller agitates the fluidic medium by stirring.
15. The stirred-tank reactor system of claim 14, wherein said
impeller is selected from the group consisting of Rushton,
hydrofoil, pitched blade, and marine.
16. The stirred-tank reactor system of claim 1, wherein said bag is
affixed to the shaft and the bearing through at least one seal or
o-ring such that the inside of the bag remains sterile.
17. The stirred-tank reactor system of claim 16, wherein said seals
or o-rings are affixed to the bag.
18. The stirred-tank reactor system of claim 16, wherein said bag
further includes a pH sensor and a dissolved-oxygen sensor, wherein
said sensors are incorporated into the bag.
19. The stirred-tank reactor system of claim 16, wherein said
system further includes at least one internal pouch sealed to the
bag, wherein the pouch includes one end that can be opened to the
outside of the bag such that a probe can be inserted into the
reactor.
20. The stirred-tank reactor system of claim 19, wherein said probe
is selected from the group consisting of a temperature probe, a pH
probe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide
sensor, a cell mass sensor, a nutrient sensor, and an
osmometer.
21. The stirred-tank reactor system of claim 16, wherein said
system further includes a least one port in the bag allowing for
the connection of a device to the port, wherein said device is
selected from the group consisting of a tube, a filter, a sampler,
a probe and a connector.
22. The stirred-tank reactor system of claim 21, wherein the port
allows for gas flow in and out of the bag.
23. The stirred-tank reactor system of claim 21, wherein the port
allows for media flow in and out of the bag.
24. The stirred-tank reactor system of claim 21, wherein the port
allows for sampling, inoculation, titration, adding of chemostat
reagents, and sparging.
25. A method for preparing a stirred-tank reactor system,
comprising: (i) providing a flexible bag with at least one opening,
wherein the bag functions as a sterile container for a fluidic
medium; (ii) inserting a shaft with an impeller attachable to said
shaft into the bag, wherein said impeller is used to agitate the
fluidic medium to provide a hydrodynamic environment; (iii)
attaching a bearing to said shaft and to said opening of the bag;
and (iv) sealing the bag to the shaft and the bearing such that the
inside of the bag remains sterile.
26. The method of claim 25, wherein said stirred-tank reactor
system is pre-sterilized.
27. The method of claim 25, wherein said stirred-tank reactor
system is disposable.
28. The method of claim 25, wherein said stirred-tank reactor
system includes at least one disposable element selected from the
group consisting of the bag, the shaft, the impeller, and the
bearing.
29. A kit comprising: (i) the stirred-tank reactor system of claim
16; and (ii) instructions for use.
30. The kit of claim 29, wherein said stirred-tank reactor system
is disposable.
31. The kit of claim 29, wherein said stirred-tank reactor system
includes at least one disposable element selected from the group
consisting of the bag, the shaft, the impeller, and the
bearing.
32. The kit of claim 29, wherein said bag is affixed to the shaft
and the bearing through at least one seal or o-ring such that the
inside of the bag remains sterile.
33. The kit of claim 29, wherein said bag further includes a pH
sensor and a dissolved-oxygen sensor, wherein said sensors are
incorporated into the bag.
34. The kit of claim 29, wherein said system further includes at
least one internal pouch sealed to the bag, wherein the pouch
includes one end that can be opened to the outside of the bag such
that a probe can be inserted into the reactor.
35. The kit of claim 34, wherein said probe is selected from the
group consisting of a temperature probe, a pH probe, a dissolved
gas sensor, an oxygen sensor, a carbon dioxide sensor, a cell mass
sensor, a nutrient sensor and an osmometer.
36. The kit of claim 29, wherein said system further includes a
least one port in the bag allowing for the connection of a device
to the port, wherein said device is selected from the group
consisting of a tube, a filter, a sampler, a probe and a
connector.
37. The kit of claim 36, wherein the port allows for gas flow in
and out of the bag.
38. The kit of claim 36, wherein the port allows for media flow in
and out of the bag.
39. The kit of claim 36, wherein the port allows for sampling,
inoculation, titration, adding of chemostat reagents, and
sparging.
40. A bag for use in the stirred-tank reactor system of claim
1.
41. The bag of claim 40, wherein said bag is a disposable plastic
bag.
42. The bag of claim 40, wherein said bag further includes at least
one disposable element selected from the group consisting of a
seal, an o-ring, a port, a pouch, a tube, a filter, a sampler, a
probe, a sensor and a connector.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a stirred-tank reactor
system and methods of preparing such systems. The present invention
further encompasses the use of the stirred-tank reactor system as a
disposable bioreactor and in kits with disposable elements.
BACKGROUND OF THE INVENTION
[0002] A bioreactor or fermenter is a container used for
fermentation, enzymatic reactions, cell culture, biologicals,
chemicals, biopharmaceuticals, tissue engineering, microorganisms,
plant metabolites, food production and the like. Bioreactors vary
in size from benchtop fermenters to stand-alone units of various
sizes. The stringent asepsis requirements for sterile production in
bioreactors usually requires elaborate systems to achieve the
desired product volumes. Consequently, the production of products
in aseptic bioreactors is costly which provides the motivation for
pursuing improved systems.
[0003] Conventional bioreactors perfuse nutrient media through a
single type of hollow fiber. The various disadvantages of such
bioreactors include heterogeneous cell mass, difficult procurement
of representative cell growth samples, poor performance due to
inefficient oxygenation and an inability to control oxygen levels,
and problems with contamination of cell cultures. Moreover,
micro-environmental factors such as pH cannot be effectively
controlled and a mixed culture or co-culture of cells is not
possible. An improvement to such prior art bioreactors is the
hollow fiber reactor, as covered in U.S. Pat. No. 5,622,857. This
reactor comprises a reaction container, through which a central
strand of porous hollow fibers extends, through which a nutrient
solution is pumped. This central strand of hollow fibers is
concentrically surrounded by a plurality of strands of hollow
fibers, through which a gaseous medium is conveyed. The hollow
fibers of these strands are also constituted in such a manner that
the gaseous medium--for example oxygen or carbon dioxide--can at
least partly emerge from these strands or enter into these strands
respectively. This type of bioreactor achieves a somewhat enhanced
nutrient media oxygenation as compared to prior art devices.
However, occasional contamination of cell cultures and an inability
to control pH levels effectively remain consistent problems.
[0004] The expense of producing cells, biopharmaceuticals,
biologicals and the like in aseptic bioreactors is exacerbated by
the required cleaning, sterilization and validation of the standard
bioreactors (i.e., stainless steel or glass reactors). Attempts
have been made to solve this problem with the development of
pre-sterilized disposable bioreactor systems that need not be
cleaned, sterilized or validated by end users. The use of such
disposable bioreactor systems could provide significant savings.
Furthermore, plastics are lightweight, easy to transport, and
require less room than stainless steel or glass reactors. An
example for the use of disposable elements in bioreactors is
provided in U.S. Pat. No. 6,245,555 B1 which describes a reactor
chamber with a support housing. The interior chamber of the support
housing is lined with a disposable liner and sealed with a head
plate attached to the liner to form a sealed chamber. Since the
liner is open at the top, it must be used in a vertically oriented
bioreactor to prevent the contamination of the head plate. Although
this system provides a disposable liner, the head plate and the
interior chamber still require cleaning and sterilization.
[0005] Another solution has been to develop flexible, disposable
plastic vessels that do not require cleaning or sterilization and
require only minimal validation efforts. For example, U.S. Pat. No.
5,523,228 describes a flexible, disposable, and gas permeable cell
culture chamber that is horizontally rotated. The cell culture
chamber is made of two sheets of plastic fused together. In
addition, the culture chamber is made of gas permeable material and
is mounted on a horizontally rotating disk drive that supports the
flexible culture chamber without blocking airflow over the membrane
surfaces. The chamber is placed in an incubator and oxygen transfer
is controlled by controlling the gas pressure in the incubator
according to the permeability coefficient of the bag. The rotation
of the bag assists in mixing the contents of the bag. However, the
cell culture chamber is limited to use within a controlled gas
environment. Particularly, the cell culture chamber has no support
apparatus and is thus limited to small volumes. Furthermore, the
chamber does not provide an inlet and an outlet for media to be
constantly pumped into and out of the chamber during rotation.
[0006] Some companies have developed a range of pre-sterile,
disposable bioreactors that do not require cleaning or sterilizing
(e.g., Wave Biotech, Bridgewater, N.J.). Such reactors are made of
sheets of flexible, gas impermeable material to form a bag. The bag
is partially filled with media and then inflated with air that
continually passes through the bag's headspace. The media is mixed
and aerated by rocking the bags to increase the air-liquid
interface. However, since there is no solid housing that support
the bags, the bags become cumbersome and difficult to handle as
they increase in size. Furthermore, the wave action within the
rocking bag creates damaging turbulent forces. Certain cell
cultures, particularly human cell cultures, develop better under
more gentle conditions.
[0007] Thus, there is a continuing need in the art to develop
flexible, pre-sterilized, disposable bioreactors that are easy to
handle and require little training to operate, yet provide the
necessary gas transfer and nutrient mixing required for successful
cell and tissue cultures. Such disposable bioreactors would be
equally useful for the production of chemicals, biopharmaceuticals,
biologicals, cells, microorganisms, plant metabolites, foods and
the like.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides a stirred-tank reactor system
with disposable elements, such as a flexible plastic bag with an
attached bearing, shaft, and impeller assembly. The instant
invention further relates to the use of this novel stirred-tank
reactor system as a disposable bioreactor and in kits with
disposable elements. The advantages of the present invention are
numerous. Particularly, the stirred-tank reactor system may be
pre-sterilized and does not require a steam-in-place (SIP) or
clean-in-place (CIP) environment for changing from batch to batch
or product to product in a culture or production system. As such,
the system requires less regulatory control by assuring zero
batch-to-batch contamination and can, thus, be operated at a
considerable cost-advantage and with minimal or no preparation
prior to use. In addition, the system is a true stirred-tank
reactor system unlike other disposable reactors systems. This
provides the added advantage that the instant invention offers a
hydrodynamic environment that can be scaled to various sizes
similar to conventional non-disposable reactor systems. Since the
system does not require cleaning or sterilizing it combines a
flexible, easy-to-use, true stirred-tank reactor environment with
zero cross-contamination during the cell culture or production
process.
[0009] One aspect of the present invention provides a stirred-tank
reactor system, comprising a flexible bag with at least one
opening, wherein the bag functions as a sterile container for a
fluidic medium; a shaft situated within the bag; an impeller
attachable to the shaft, wherein the impeller is used to agitate
the fluidic medium to provide a hydrodynamic environment; and a
bearing attached to the shaft and to the opening of the bag. The
bag may be affixed to the shaft and the bearing through at least
one seal or o-ring such that the inside of the bag remains sterile.
The seals or o-rings can be affixed to the bag. The system may be
disposable and pre-sterilized. The bag may further include a pH
sensor and a dissolved-oxygen sensor, wherein the sensors are
incorporated into the bag. In addition, the system may include at
least one internal pouch sealed to the bag, wherein the pouch has
one end that can be opened to the outside of the bag such that a
probe (i.e., a temperature probe, a pH probe, a dissolved gas
sensor, an oxygen sensor, a carbon dioxide (CO.sub.2) sensor, a
cell mass sensor, a nutrient sensor, an osmometer, and the like)
can be inserted into the reactor. The system may also include at
least one port in the bag allowing for the connection of a device
such as a tube, a filter, a sampler, a probe, or a connection
device to the port. A port allows for sampling; gas flow in and out
of the bag; liquid or media flow in and out of the bag;
inoculation; titration; adding of chemostat reagents; sparging; and
the like.
[0010] Another aspect of the present invention provides a
stirred-tank reactor system, comprising a flexible bag with at
least one opening, wherein the bag functions as a sterile container
for a fluidic medium; a shaft situated within the bag; an impeller
attachable to the shaft, wherein the impeller is used to agitate
the fluidic medium to provide a hydrodynamic environment; and a
bearing attached to the shaft and to the opening of the bag. The
system may further include a housing, such as a reactor housing, on
the outside of the bag, wherein the housing includes at least one
support that holds the bearing and a motor, and wherein the bag is
contained within the housing. The housing may further include a
plurality of baffles such that the bag folds around the baffles.
Optionally, the system further encompasses a heater (e.g., a
heating pad, a steam jacket, a circulating fluid or water heater,
etc.) that can be located between the bag and the housing.
Alternatively, the heater may be incorporated into the housing
(e.g., a permanent reactor housing with incorporated heating
system).
[0011] In another aspect of the invention, the stirred-tank reactor
system includes a permanent housing with a product loop with flow
past a pH sensor and a dissolved-oxygen sensor, wherein the sensors
are incorporated into the housing. The permanent housing includes,
but is not limited to, a metal barrel, a plastic barrel, a wood
barrel, a glass barrel, and the like.
[0012] The invention also contemplates a method for preparing a
stirred-tank reactor system, comprising providing a flexible bag
with at least one opening, wherein the bag functions as a sterile
container for a fluidic medium; inserting a shaft with an impeller
attachable to the shaft into the bag, wherein the impeller is used
to agitate the fluidic medium to provide a hydrodynamic
environment; attaching a bearing to the shaft and to the opening of
the bag; and sealing the bag to the shaft and the bearing such that
the inside of the bag remains sterile. The stirred-tank reactor
system prepared by this method includes at least one disposable
element including, but not limited to, the bag, the shaft, the
impeller, and the bearing.
[0013] The invention further encompasses a kit comprising a
stirred-tank reactor system and instructions for use. The kit
includes a disposable stirred-tank reactor system. The kit may also
include a stirred-tank reactor system with at least one disposable
element such as the bag, the shaft, the impeller, or the bearing.
The bag may be affixed to the shaft and the bearing through at
least one seal or o-ring such that the inside of the bag remains
sterile. Furthermore, the bag may include a pH sensor and a
dissolved-oxygen sensor, wherein the sensors are incorporated into
the bag. The kit may also include at least one internal pouch
sealed to the bag, wherein the pouch includes one end that can be
opened to the outside of the bag such that a probe can be inserted
into the reactor. In addition, the system may include at least one
port in the bag allowing for the connection of a device to the
port, wherein the device includes, but is not limited to, a tube, a
filter, a sampler, and the like.
[0014] Another aspect of the invention provides a bag for use in a
stirred-tank reactor system. The bag may be a disposable, flexible,
plastic bag. The bag may also include at least one disposable
element including, but not limited to, a seal, an o-ring, a port, a
pouch, a tube, a filter, a sampler, a probe, a sensor, a connection
device, or the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention is best understood when read in
conjunction with the accompanying figures which serve to illustrate
the preferred embodiments. It is understood, however, that the
invention is not limited to the specific embodiments disclosed in
the figures.
[0016] FIG. 1 depicts a longitudinal cross-section of one
embodiment of the stirred-tank reactor system, wherein the
stirred-tank reactor system is placed into a permanent housing.
[0017] FIG. 2 depicts a probe connection in order to illustrate
that a probe can be attached to the stirred-tank reactor system via
a sterile or aseptic connection.
DETAILED DESCRIPTION OF THE INVENTION
[0018] a) Definitions and General Parameters
[0019] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0020] The term "flexible bag" refers to a container that holds a
fluidic medium. The bag may include one or more layer(s) of
flexible or semi-flexible waterproof material depending on size,
strength and volume requirements. The inside surface of the bag is
preferably smooth and provides a sterile environment (e.g., for
culturing cells or other organism, for food production, etc.). The
bag may include one or more openings, pouches (e.g., for inserting
one or more probes, devices, etc.), ports (e.g., for the connection
of one or more probes, devices, etc.) or the like. Furthermore, the
bag provides a disposable alternative to a solid vessel in a
conventional stirred-tank bioreactor. The flexible bag may further
include a shaft, an impeller, a bearing and seals or o-rings, and
may be entirely disposable.
[0021] The term "fluidic medium" means, for the purpose of the
specification and claims, any biological fluid, cell culture
medium, tissue culture medium, culture of microorganisms, culture
of plant metabolites, food production, chemical production,
biopharmaceutical production, and the like. The fluidic medium is
not limited to any particular consistency and its viscosity may
vary from high to medium to low. When the fluidic medium is a cell
culture medium the system may be operated in batch mode, semi-batch
mode, fed-batch mode, or continuous mode.
[0022] The term "impeller" refers to a device that is used for
agitating or mixing the contents of a stirred-tank reactor system
(e.g., bioreactor). The impeller may agitate the fluidic medium by
stirring or other mechanical motion. The impeller of the instant
invention includes, but is not limited to, a Rushton, a marine, a
hydrofoil, a pitched blade, and any other commercially available
impeller.
[0023] A "hydrodynamic" environment of the instant invention refers
to an environment that is influenced by the motion of fluids and
the forces acting on solid bodies immersed in these fluids within
the stirred-tank reactor system.
[0024] b) The Stirred-Tank Reactor System
[0025] The stirred-tank reactor system of the present invention
provides a flexible and disposable bag for a variety of purposes,
including culturing cells, microorganisms, or plant metabolites as
well as processing foods, chemicals, biopharmaceutical and
biologicals. The disposable bag may include disposable elements
such as a shaft, impeller and bearing and is designed to fit into a
permanent housing such as a reactor housing. The bag may further
include one or more openings, pouches, ports or the like. The
stirred-tank reactor system allows a user to operate the culture or
production with relative ease and little training. In particular,
the disposable system does not require cleaning or sterilizing.
Furthermore, the system does not need continuous validation between
production runs. Thus, it combines a flexible, easy-to-use, true
stirred-tank reactor environment with zero cross-contamination
during the production process.
[0026] Referring to the drawings, FIG. 1 depicts a flexible bag (4)
with at least one opening and an agitation shaft (12) with an
attachable impeller (13). As shown, the agitation shaft (12) and
attached impeller (13) are situated within the bag (4). Further,
the agitation shaft (12) is connectable to a bearing (5), wherein
the bearing can be sealed to the bag through seal(s) or o-ring(s)
(6). The bag (4), agitation shaft (12), impeller (13), and bearing
(5), including seals or o-rings (6) are optionally disposable. The
disposable bag can be a flexible, plastic bag. The bag (4) can be
affixed to the agitation shaft (12) and the bearing (5) through at
least one seal or o-ring (6) such that the inside of the bag
remains sterile. The seals or o-rings can be further affixed to the
bag as is shown in FIG. 1. Additionally, the disposable
stirred-tank reactor system may be connected to a support or one or
more bracket(s) (3) that hold the bearing (5) and motor (1). In one
embodiment (as shown in FIG. 1), the support (3) is a motor and
bearing support (3), wherein the upper end of the agitation shaft
(12) is further connected to a motor coupling (2). The motor
coupling (2) is connected to the motor (1) which drives the
stirring motion of the agitation shaft (12) and impeller (13)
leading to a hydrodynamic environment within the bag (4). The bag
(4) is designed to fit into a housing (11) such as a barrel or
chamber. The housing may be a metal barrel, a plastic barrel, a
wood barrel, a glass barrel, or any other barrel or chamber made
from a solid material. In one embodiment of the instant invention,
the housing further includes a plurality of baffles, wherein the
bag folds around the baffles. In another embodiment, the flexible
bag (4) further includes a top port (single or multiple) (8), a
bottom port (single or multiple) (9), and a side port (single or
multiple) (10), wherein flexible tubing (7) can be connected to one
or more of these ports.
[0027] The stirred-tank reactor system may optionally include a
heater such as a heating pad, a steam jacket, or a circulating
fluid or water heater. In one embodiment, the heater is located
between the bag (4) and the housing (11). In another embodiment,
the heater is incorporated into the housing (11) (e.g., into a
double wall between the reactor housing and the bag). In yet
another embodiment, the stirred-tank reactor system is placed
inside an incubator. The heater allows for heating or warming of a
specific culture or production. This is particularly important for
cell cultures which are often grown at 37.degree. C.
[0028] In one embodiment of the instant invention, the bag (4), the
bearing (5), the seal(s) or o-ring(s) (6), the tubing (7), the top
port(s) (8), the bottom port(s) (9), the side port(s) (10), the
shaft (12), and the impeller (13) are disposable. The motor (1),
the motor coupling (2), the bracket(s) or motor and bearing support
(3), and the housing (11) are permanent.
[0029] c) Devices and Ports
[0030] The stirred-tank reactor system may also include sensors and
other devices. In one embodiment, the bag includes a pH sensor and
a dissolved-oxygen sensor, wherein the sensors are incorporated
into the bag. As such, the sensors are disposable with the bag. In
another embodiment, the sensors are attachable to the bag and are
separate units. Such sensors may optionally be reusable after
sterilization. In another embodiment, the system includes a product
loop with flow past a pH sensor and dissolved-oxygen sensor,
wherein the sensors are incorporated into the reactor housing. The
system is flexible and provides alternative ways of supplying
optional equipment of various kinds (e.g., sensors, probes,
devices, pouches, ports, etc.). The system may also include one or
more internal pouches that are sealed to the bag. In one preferred
embodiment, the pouch has at least one end that can be opened to
the outside of the bag to insert a probe into the reactor (i.e.,
the bag) while remaining on the exterior of the bag. The probe may
be, for example, a temperature probe, a pH probe, a dissolved gas
sensor, an oxygen sensor, a carbon dioxide sensor, a cell mass
sensor, a nutrient sensor, an osmometer or any other probe that
allows for testing or checking the culture or production. In
another preferred embodiment, the system includes at least one port
in the bag allowing for the connection of a device to the port.
Such a device includes, but is not limited to, a tube, a filter, a
connector, a probe, and a sampler. The incorporation of various
ports into the bag allows for gas flow in and out of the bag as
well as liquid flow in and out of the bag. Such ports also allow
for sampling or testing the media or culture inside the bag.
Tubing, filters, connectors, probes, samplers or other devices can
be connected to the ports by using any desirable tubing connection
technology. Pouches and ports that are sealed or affixed to the bag
are disposable with the bag. The bag may also include a sparger
(i.e., the component of a reactor that sprays air into the medium)
sealed to the bag which can be disposed off with the bag.
[0031] Particularly, ports may be incorporated at any place on the
flexible bag to accommodate the following:
[0032] Headspace gas in
[0033] Headspace gas out
[0034] Sparge gas in
[0035] Temperature probe
[0036] pH probe
[0037] Dissolved oxygen probe
[0038] Other desired probes
[0039] Sample apparatus
[0040] Media in
[0041] Titrant in
[0042] Inoculum in
[0043] Nutrient feeds in
[0044] Harvest out
[0045] Each port may have flexible tubing attached to the port, to
which media bags, sample devices, filters, gas lines, or harvest
pumps may be attached with sterile or aseptic connections. In one
embodiment, the ports are sealed onto the flexible bag during bag
manufacture, and are sterilized with the bag assembly.
[0046] Devices that may be used to make aseptic connections to the
flexible tubing are the following:
[0047] WAVE sterile tube fuser
[0048] TERUMO sterile tubing welder
[0049] PALL KLEENPAK connector
[0050] Connection made under a laminar flow hood, using aseptic
techniques
[0051] BAXTER Hayward proprietary "HEAT-TO-HEAT" connection using
metal tubing and an induction heater
[0052] In another embodiment, flexible tubing that is attached to
an appropriate stainless-steel valve assembly may be sterilized
separately (e.g., via autoclave), and then used as a way to connect
the disposable bioreactor to traditional reactors or process
piping. The valve assembly is used to make a traditional
steam-in-place (SIP) connection to a traditional reactor or other
process, and the flexible tubing is used to make a sterile or
aseptic connection to a port on the disposable reactor.
[0053] Referring to the drawings, FIG. 2 depicts a probe connection
that can be employed with the stirred-tank reactor system of the
instant invention. In one embodiment (as shown in FIG. 2), the
probe (1) is connected to a flexible sleeve (2) or bag which
extends to one half of a PALL connector (3). The PALL connector (3)
can be connected to the other half of the PALL connector (5) to
provide for a sterile connection between the probe and the
stirred-tank reactor system. The PALL connectors (3), (5) include
covers (4) and filters (7) to keep the connection site sterile.
Sterile tubing (6) extends from the other half of the PALL
connector (5) to a reactor port (8) of the reactor vessel (9) of
the stirred-tank reactor system. In order to attach the probe, the
PALL connection is made by removing the covers (4), mating the
connectors (3, 5), removing the filters (7), and sliding the
movable part of the connector into position. The probe sensor tip
(12) is then pushed into the reactor as the flexible sleeve or bag
bunches or compresses (10). The probe senor tip (12) is then in
direct contact with the inside of the reactor vessel (9). A clamp
(11) is placed around the probe and tubing to seal the reactor
contents from the PALL connection assembly. Thus, when a sterile
connection is made between the two halves of the PALL connectors
(3, 5), the flexible sleeve (2) or bag becomes compressed (10) and
the probe is in contact with the culture or production media.
[0054] In one embodiment, the probes may be sterilized separately
(e.g., via autoclave) then attached to the reactor via a sterile or
aseptic connection. For example, a probe assembly may be made by
inserting a probe (1) into one half of a PALL KLEENPAK connector
(3) and sealing the probe to the connector using a flexible sleeve
or bag (2) as described above and shown in FIG. 2. The sleeve
extends from the outside end of the probe to the barb of the PALL
connector. This assembly is sterilized separately. The other half
of the PALL connector (5) is connected to a port (8) on the reactor
(9) via flexible tubing (6) that will accommodate the probe. This
assembly is sterilized as part of the reactor. The PALL connector
is described in detail in U.S. Pat. No. 6,655,655 and incorporated
herein by reference in its entirety.
[0055] d) Cultures
[0056] The stirred-tank reactor system is designed to hold a
fluidic medium such as a biological fluid, a cell culture medium, a
culture of microorganisms, a food production, or the like. When the
fluidic medium is a cell culture the system can be operated in
batch-mode, semi-batch mode, fed-batch mode, or continuous mode. A
batch culture is a large scale cell culture in which a cell
inoculum is cultured to a maximum density in a tank or fermenter,
and harvested and processed as a batch. A fed-batch culture is a
batch culture which is supplied with either fresh nutrients (e.g.,
growth-limiting substrates) or additives (e.g., precursors to
products). A continuous culture is a suspension culture that is
continuously supplied with nutrients by the inflow of fresh medium,
wherein the culture volume is usually constant. Similarly,
continuous fermentation refers to a process in which cells or
micro-organisms are maintained in culture in the exponential growth
phase by the continuous addition of fresh medium that is exactly
balanced by the removal of cell suspension from the bioreactor.
Furthermore, the stirred-tank reactor system can be used for
suspension, perfusion or microcarrier cultures. Generally, the
stirred-tank reactor system can be operated as any conventional
stirred-tank reactor with any type of agitator such as a Rushton,
hydrofoil, pitched blade, or marine. The agitation shaft (12) can
be mounted at any angle or position relative to the housing (11),
such as upright centered, upright offset, or 15.degree. offset. The
control of the stirred-tank reactor system can be by conventional
means without the need for steam-in-place (SIP) or clean-in-place
(CIP) control. In fact, the system of the instant invention is not
limited to sterile bioreactor operation, but can be used in any
operation in which a clean product is to be mixed using a stirred
tank, for example, food production or any clean-room mixing without
the need for a clean-room.
[0057] e) The Kit
[0058] The invention encompasses a kit that includes a stirred-tank
reactor system and instructions for use. In a preferred embodiment,
the kit includes a disposable stirred-tank reactor system.
Accordingly, the kit includes at least one disposable element such
as the bag, the shaft, the impeller, or the bearing. Preferably,
the kit is entirely disposable. The flexible, disposable bag may be
affixed to the shaft and the bearing through at least one seal or
o-ring such that the inside of the bag remains sterile. In
addition, the bag may include a pH sensor and a dissolved-oxygen
sensor, wherein the sensors are incorporated into the bag and are
disposable with the bag. The kit may also include one or more
internal pouches that are sealed to the bag. The pouch has one end
that can be opened to the outside of the bag such that a probe can
be inserted into the reactor. The probe may be a temperature probe,
a pH probe, a dissolved gas sensor, an oxygen sensor, a carbon
dioxide (CO.sub.2) sensor, a cell mass sensor, a nutrient sensor,
an osmometer, and the like. Furthermore, the system may include at
least one port in the bag allowing for the connection of a device
to the port, wherein the device includes, but is not limited to, a
tube, a filter, a sampler, a probe, a connector, and the like. The
port allows for sampling, titration, adding of chemostat reagents,
sparging, and the like. The advantage of this kit is that it is
optionally entirely disposable and easy-to-use by following the
attached instructions. This kit comes in different sizes depending
on the preferred culture volume and can be employed with any
desired reaction chamber or barrel. This kit is pre-sterilized and
requires no validation or cleaning. The kit can be used for cell
culture, culture of microorganisms, culture of plant metabolites,
food production, chemical production, biopharmaceutical production,
and others.
[0059] In another embodiment the kit includes a housing or barrel
that holds the disposable bag. Such a housing or barrel can be
supplied with the bag or provided separately.
[0060] f) Examples
[0061] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims.
[0062] (i) A Disposable Bioreactor
[0063] One example of a stirred-tank reactor system of the instant
invention is a disposable bioreactor. The bioreactor is similar to
a 600 liter media bag with built-in agitation and attachable
sensors (e.g., pH sensors, temperature sensors, dissolved oxygen
(dO2) sensors, etc.). The reactor is operated via conventional
controllers. The agitator (e.g., agitation shaft and impeller) and
bearing are disposable and built into the bag. The motor attaches
to a support (e.g., motor and bearing support) or bracket(s) on the
600 liter barrel that holds the bag. In size, shape, and operation,
this bioreactor appears similar to a stainless steel reactor with a
sterile liner, however, the bioreactor of this invention provides a
multitude of advantages compared to a conventional stainless steel
reactor. Most importantly, the need for cleaning and steam
sterilization is eliminated. The bag is pre-sterilized by
irradiation and, thus, ready for use. In fact, no cleaning,
sterilization, validation or testing is required at culture
start-up or between culture runs. Consequently, the bioreactor
provides a culture environment of zero cross-contamination between
runs. In conventional systems, the majority of costs are related to
clean-in-progress (CIP) and steam-in-progress (SIP) as well as the
design of a skid and control system to oversee these functions.
These costs are eliminated in the disposable bioreactor and
multiple products may be cultured or manufactured simultaneously
and with much greater ease.
[0064] The disposable bioreactor can be easily scaled-up by using
larger culture bags and larger barrels to hold the bags. Multiple
bioreactors can be operated at the same time without any need for
extensive engineering or cleaning. The bioreactor is a true stirred
tank with well characterized mixing. As such, the bioreactor has
the added advantage that it can be scaled and its contents
transferred to a stainless steel reactor if desired. Notably, the
bioreactor combines ease of use with low cost and flexibility and
provides, thus, a new technical platform for cell culture.
[0065] (ii) Cell Culture
[0066] The disposable bioreactor of the instant invention can be
used for a batch culture in which cells are inoculated into fresh
media. As the cells grow, they consume the nutrients in the media
and waste products accumulate. For a secreted product, when the
culture has run its course, cells are separated from the product by
a filtration or centrifugation step. For viral-vector production,
cells are infected with a virus during the growth phase of the
culture, allowing expression of the vector followed by harvest.
Since there is zero cross-contamination in the bioreactor it works
well with batch cultures.
[0067] The bioreactor can also be used for perfusion cultures,
wherein product and/or waste media is continuously removed and the
volume removed is replaced with fresh media. The constant addition
of fresh media, while eliminating waste products, provides the
cells with the nutrients they require to achieve higher cell
concentrations. Unlike the constantly changing conditions of a
batch culture, the perfusion method offers the means to achieve and
maintain a culture in a state of equilibrium in which cell
concentration and productivity may be maintained in a steady-state
condition. This can be accomplished in the disposable bag as easily
as in any conventional stainless steel reactor. For viral-vector
production, the perfusion process allows for an increase in the
cell concentration and, thereby the post-infection virus titer. For
a secreted product, perfusion in the bioreactor offers the user the
opportunity to increase the productivity by simply increasing the
size of the culture bag. Most importantly, there is no need for
sterilization, validation, or cleaning because the system
experiences zero cross-contamination during the production
process.
[0068] Various modifications and variations of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention which are obvious to those skilled in the art are
intended to be within the scope of the claims.
* * * * *