NZ614364B2 - Pneumatic alternating pressure membrane cell separation system - Google Patents
Pneumatic alternating pressure membrane cell separation system Download PDFInfo
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- NZ614364B2 NZ614364B2 NZ614364A NZ61436412A NZ614364B2 NZ 614364 B2 NZ614364 B2 NZ 614364B2 NZ 614364 A NZ614364 A NZ 614364A NZ 61436412 A NZ61436412 A NZ 61436412A NZ 614364 B2 NZ614364 B2 NZ 614364B2
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Abstract
614364 The disclosure provides a filtration system that can be referred to as Pneumatic Alternating Cell Separator (PACS), useful components thereof in the form of assemblies or kits of parts that can be used to build the system, and use of the system for filtering fluids, for instance in cell culture perfusion systems comprising a filter containing chamber (8), an expansion chamber (17) and a gas flow controller (28). ure perfusion systems comprising a filter containing chamber (8), an expansion chamber (17) and a gas flow controller (28).
Description
Title: Pneumatic Alternating Cell Separation (PACS) system
The present invention relates to filtration systems. More specifically, the invention
relates to a Pneumatic Alternating Cell Separation (PACS) system for fluids,
particularly biological fluids comprising cells.
Background
Filtration is typically performed to separate, clarify, modify and/or concentrate a fluid
solution, mixture or suspension. In the biotechnology and pharmaceutical industries,
filtration is vital for the successful production, processing, and testing of new drugs,
diagnostics and other biological products. For example, in the process of
manufacturing biologicals, using animal cell culture, filtration is done for
clarification, selective removal and concentration of certain constituents from the
culture media or to modify the media prior to further processing. Filtration may also
be used to enhance productivity by maintaining a culture in perfusion at high cell
concentration. The invention provides an improved means for fractionating a mixture
or suspension of molecules or particulates based on physical and/or chemical
properties.
Several specialized filters and filtration methods have been developed to
separate materials according to their chemical and physical properties. Filters which
have been developed in the art include flat surface filters, pleated filters, multi-unit
cassettes, and tubular forms such as hollow fibers. However, many of these filters
have short operating lives, and when used to filter cell culture suspension or other
biological fluids they tend to clog with dead cells, cell debris, aggregates or other
constituents of the fluid.
Animal cells grow substantially slower than most microorganisms, and lacking
protective cell wall, they are also more fragile. Some known methods for increasing
the productivity of microbial culture production including increasing agitation rates
and vigorous delivery of gases into the culture are not feasible with animal cells.
Thus, production is limited to very gentle culture conditions and low cell
concentrations. One way to increase the cell concentration, yet maintaining gentle
culture conditions is through the perfusion method.
In the perfusion method for growing cells, culture medium, whose nutrients
have been consumed and which contains increased levels of harmful waste products,
is continuously removed from the culture and replaced with fresh medium. The
constant addition of fresh medium while eliminating waste products provides the cells
with the nutrients required to achieve high cell concentrations. Unlike the constant
changing conditions during batch culture method of production, the perfusion method
offers the means to achieve and maintain a culture in steady state.
In normal batch cultures production processes, cells are first inoculated into a
fresh medium and the cells rapidly enter a log grow phase. As they consume the
medium nutrients and waste products accumulate, the cells transition to a stationary
followed by a decay phase. While several methods have been developed to optimize
batch culture production, in each case, these processes undergo rapid growth and
decay cycles. In perfusion, however, since waste products generated by the culture are
continuously removed and the culture is continuously replenished with fresh medium,
it is possible to achieve a state of equilibrium in which cell concentration and
productivity are maintained. Typically, about one culture volume is exchanged per
day and the cell concentration achieved in perfusion are typically 2 to more than 10
times that achieved at the peak of batch culture.
Filtration systems for biological fluids were described previously in the art.
One type of external filtration perfusion systems is for instance described in US Patent
6,544,424, which is incorporated herein by reference. This fluid filtration system
comprises a fluid storage vessel connected to a filter containing compartment that is
connected to a diaphragm pump. The diaphragm pump alternatively aspirates the fluid
out of the vessel through the filter and expels the fluid through the retentate end of the
filter, back into the vessel. By doing so, the system creates an alternating tangential
flow of fluid through the filter element.
A major drawback of said system is that the diaphragm pump contains moving
parts which are prone to wastage and can often break during the process. When the
diaphragm breaks the filtration system becomes no longer closed and becomes
susceptible for contamination. The filtration process must therefore be aborted,
leading to high costs in the case of e.g. processes for production of
biopharmaceuticals. Indeed, since processes for the production of biopharmaceutical
molecules take several days sometimes up to several weeks, the breakage of a pump
during a process run would lead to high costs and long down time in a production
facility. It takes up to days for getting a new process running. These perfusion cultures
are mainly performed at the last stage of the production process which means that a
lot of time and money is wasted due to this failure. It takes usually 3 to 5 weeks
before the stage of perfusion is reached. The costs involved can easily increase to
more than 100.000 Euros.
A second drawback is that the diaphragm pump described in US 6,544,424
consists of a stainless steal jacket which contains a diaphragm usually made of rubber
or silicone. Before each run, the elements of said diaphragm pump must be cleaned,
assembled and sterilized. In addition, when the system is used for production of
pharmaceutical products, said cleaning and sterilization procedures must be validated.
Said validation implies lengthy procedures and test runs, which are very costly and
time consuming.
A third drawback of the systems currently in use is that they use invasive
sensor technology wherein sensors are in contact with the product and wherein
sensors cannot be replaced during the process.
The present invention aims at providing improved fluid filtration systems that
have less of or eliminate these drawbacks, and/or at least provide the public with a
useful choice.
Summary of the invention
The term ‘comprising’ as used in this specification and claims means
‘consisting at least in part of’. When interpreting statements in this specification and
claims which include the term ‘comprising’, other features besides the features
prefaced by this term in each statement can also be present. Related terms such as
‘comprise’ and ‘comprised’ are to be interpreted in a similar manner.
The disclosure provides a novel filtration system that can be referred to as
Pneumatic Alternating Cell Separator (PACS), useful components thereof in the form
of assemblies or kits of parts that can be used to build the system, and use of the
system for filtering fluids, for instance in cell culture perfusion systems. The present
disclosure provides a filtration system comprising a vessel connected to an entrance
end of a filter containing compartment which at the exit end is connected to an
expansion chamber, which is connected to a gas flow controller. Said gas flow
controller alternately provides for positive and negative pressure into the expansion
chamber allowing for the fluid contained in the storage vessel to be alternately
aspirated through the filter element into the expansion chamber and expelled from the
expansion chamber, through the filter, back into the vessel. By doing so, the system
creates an alternating tangential flow of fluid through the filter element. Said fluid
flow can further be guided through a fluid harvest port into a recovery container. The
system is useful for conducting a rapid, low shear, tangential flow filtration. Such a
system has applications in perfusion of cultured animal cells as well as other varied
filtration applications.
The system of the present disclosure offers the benefits of tangential flow filtration
without some of its drawbacks. As will become apparent, some of the benefits not
offered by other systems in the art include the absence of moving parts, which are
required in similar systems to provide for the alternating liquid flow. Indeed, certain
systems known in the art comprise a diaphragm pump that alternatively aspirates the
fluid out of the vessel through the filter containing compartment and expels the fluid
through the retentate end of the filter, back into the vessel. Said diaphragm, which is a
fragile rubber membrane, can easily rupture when extensively used.
The system of the present disclosure does not require a diaphragm pump that
can easily break, nor any moving parts that are prone to wastage due to extensive use
of the system. The system of the present disclosure comprises an expansion chamber,
with at least two openings, which is connected on one side (first opening) to the exit
end of a filter containing compartment allowing for liquid from said compartment to
enter the chamber and on the other side (second opening) to a gas flow controller
which alternately provides negative and positive pressure into the expansion chamber
thereby aspirating fluid from the exit end of the filter containing compartment and
expelling fluid back into the exit end of the filter containing compartment.
Herewith, the expansion chamber in the present system comprises a direct gas-
liquid interface without separation means, said interface being formed by the liquid
contained in the system which is in direct contact with the gas phase provided by the
gas flow controller. The expansion chamber in the present disclosure does not contain
any separation means between the first and second opening of the chamber. The
expansion chamber comprises one room or space and is not separated into two or
more compartments. The absence of separation means such as a diaphragm or any
other moving parts increases the life time of the system and minimizes the risks of
premature abortion of a process cycle.
In addition, the filtration system according to preferred embodiments of the
present invention does not require costly cleaning and sterilization validation. Instead
it comprises in such embodiments disposable elements which are pre-sterilized and
ready for use. This offers a great advantage considering validation costs and assembly
time.
In further preferred embodiments, a third remarkable advantage of the present
system is that it uses non invasive sensors. The filtration system according to the
present disclosure requires sensors that are not in contact with product and which can
be replaced –in process- when broken. This allows for a more controlled process
environment during the whole length of the process run.
The invention provides for a fluid filtration system comprising:
a) at least one fluid storage vessel;
b) at least one filter containing compartment;
c) at least one fluid transfer line connecting the storage vessel to an entrance end of a
filter containing compartment, which fluid transfer line is capable of directing a fluid
from the storage vessel into the entrance end of the filter containing compartment;
d) at least one expansion chamber with at least two openings,
wherein a first opening is connected at an exit end of the filter containing
compartment and wherein a second opening is connected to a gas flow controller,
wherein the expansion chamber contains no separation means between the first and
second opening ; and wherein at least one level sensor is mounted on or in the
expansion chamber
e) at least one gas flow controller for alternately providing the expansion chamber
with positive and negative pressure;
f) at least one fluid harvest port connected to the filter containing compartment for
removing filtered fluid from the filter containing compartment.
In one embodiment the expansion chamber is connected to the gas flow
controller with a gas line. In one embodiment said gas line comprises a sterile filter
between the expansion chamber and the gas flow controller. In one embodiment said
sterile filter is provided with a heater.
In one embodiment of the present invention, the fluid storage vessel is a
bioreactor.
In one embodiment the filter containing compartment contains a hollow fiber
filter. In another embodiment the filter containing compartment contains a screen
filter.
In yet another embodiment the fluid filtration system according to the
invention comprises at least one level sensor mounted on the surface of the expansion
chamber.
In a preferred embodiment the fluid filtration system comprises at least one
level sensor mounted on or in the expansion chamber, preferably wherein at least two
level sensors are mounted on or in the expansion chamber, and preferably wherein the
level sensor is or level sensors are mounted on the surface (i.e. outside) of the
expansion chamber.
In one embodiment, said level sensors are capable of measuring a minimal and
a maximal fluid level in the expansion chamber, and are functionally coupled to the
gas flow controller.
In one embodiment the fluid filtration system according to the disclosure is
capable of alternately aspirating the fluid of the system from the storage vessel,
through the filter containing compartment into the expansion chamber, and expelling
the fluid from said expansion chamber through the filter containing compartment back
into the storage vessel. Fluid aspiration is performed by applying negative pressure
into the expansion chamber and fluid expulsion is performed by applying positive
pressure into the expansion chamber.
Preferably, said negative pressure is obtained by creating a vacuum in the
expansion chamber and positive pressure is obtained by injecting gas, for instance
compressed air, into the expansion chamber.
In one preferred embodiment the fluid filtration system according to the
present disclosure comprises a) at least one fluid storage vessel;
b) at least one filter containing compartment; wherein the filter comprises a plurality
of bundled hollow fibers whose axes extend longitudinally from the entrance end to
the exit end of the filter containing compartment.
c) a fluid transfer line connected at one end thereof to the storage vessel, and attached
at another end thereof to an entrance end of the filter containing compartment, which
fluid transfer line is capable of directing a fluid from the storage vessel into the
entrance end of the filter containing compartment;
d) at least one expansion chamber connected at a retentate exit end of the filter
containing compartment; which expansion chamber is capable of alternately
aspirating fluid from the retentate exit end of the filter containing compartment and
expelling the fluid back into the retentate exit end of the filter containing
compartment; said expansion chamber being connected to a gas flow controller
capable of alternately applying negative and positive pressure into the expansion
chamber and thereby forming a direct gas-liquid interface without separation means
into the expansion chamber; and
e) at least one fluid harvest port connected to the filter containing compartment for
removing filtered fluid from the permeate exit end of the filter containing
compartment, said harvest port optionally being connected via a permeate line to a
fluid pump.
In certain embodiments, the filter containing compartment and/or the
expansion chamber are disposable, i.e. made of disposable materials, and in further
preferred embodiments also the fluid storage vessel and/or the fluid transfer line are
disposable. Preferably, the complete fluid filtration system is disposable.
Another aspect of the disclosure relates to an assembly comprising an
expansion chamber assembled to a filter containing compartment on one side and to
an air filter on the other side. Preferably, said assembly is capable of being
functionally coupled to a gas line that can provide positive and negative pressure to
the expansion chamber. Preferably the assembled components exclusively comprise
disposable materials.
Another aspect of the present disclosure relates to a kit of parts comprising an
expansion chamber, a filter containing compartment and an air filter, intended for
providing an assembly as described herein.
The present invention also provides a process for filtering a fluid comprising
a) providing a fluid filtration system comprising at least one fluid storage vessel; at
least one filter containing compartment; a fluid transfer line connecting the storage
vessel to an entrance end of the filter containing compartment, which transfer line is
capable of directing a fluid from the storage vessel into the entrance end of the filter
containing compartment;
at least one expansion chamber connected on one side to an exit end of the
filter containing compartment allowing for liquid from said compartment to enter the
chamber and on the other side to a gas flow controller which alternately provides
negative and positive pressure into the expansion chamber thereby aspirating fluid
from the exit end of the filter containing compartment and expelling fluid back into
the exit end of the filter containing compartment, forming a direct gas-liquid interface
without separation means into the expansion chamber;
and at least one permeate port connected to the filter containing compartment
for removing filtered fluid from the filter containing compartment; b) drawing fluid
out of the storage vessel through the filter into the expansion chamber by applying
negative pressure into the expansion chamber; c) expelling the fluid from the
expansion chamber through the filter back into the storage vessel by applying positive
pressure into the expansion chamber; d) repeating steps b and c generating an
alternative tangential flow of fluid through the filter; and e) removing the filtered fluid
from the filtration system.
The disclosure also provides a process for filtering a fluid according to the
disclosure, wherein the process is performed using a fluid filtration system according
to the disclosure.
In a preferred embodiment of said process, the filter containing compartment
and/or the expansion chamber are disposable. Preferably the filter containing
compartment, the expansion chamber, the fluid storage vessel and the fluid transfer
line are disposable.
In another embodiment, the positive and negative pressure, during the process,
are regulated by level sensors which measure the fluid level in the expansion
chamber. Said level sensors are preferably mounted on the expansion chamber.
Description of the drawings
Fig. 1 shows a first embodiment of an alternating tangential flow filtration system
according to the invention showing a harvest port at the bottom side of the vessel
connected to the bottom side part of a filter compartment.
Fig. 2 shows an assembly comprising an expansion chamber, a filter containing
compartment and an air filter.
Fig. 3 shows a cell growth curve in a 10L bioreactor coupled to a PACS system.
Fig. 4 shows a cell growth curve in a 10L bioreactor coupled to a PACS system.
Detailed description
The present invention concerns a fluid filtration system generally comprising at least
one fluid storage vessel, a fluid transfer line for directing fluid from the vessel through
a filter containing compartment, at least one expansion chamber connected to the filter
containing compartment on one side by means of a fluid connector and to a gas flow
controller on the other side by means of a gas line, and at least one fluid harvest port
coming out of the filter containing compartment. Said gas flow controller alternatively
provides for vacuum or compressed air into the expansion chamber in order to power
the fluid in alternating directions through the filter containing compartment. Herewith,
a direct gas-liquid interface is formed in the expansion chamber between the fluid in
the system and the compressed air provided by the gas flow controller. A separation
means between the liquid and gas phase is not required in the present system contrary
to systems known in the art, which for example comprise a diaphragm pump that
expels and aspirates the fluid in and out of the filter containing compartment. The
absence in the present invention of a diaphragm pump or other moving parts increases
the life time of the system and minimizes the risks of premature abortion of a process
cycle, as a diaphragm pump or other moving parts can easily break.
The system offers a gentle treatment of fragile materials such as animal cells
and biomolecules. A uniform flow can be generated across the entire filter, herewith
providing a means for generating rapid, low shear, tangential flow. The system has
applications in perfusion of cultured animal cells as well as other varied filtration
applications. Hollow fiber (HF) type filters afford longer operating lives, and they are
available in many sizes, configurations, materials, pore sizes and porosity.
Furthermore, the process need not be limited to the use of hollow fiber filters. It is
possible to insert other separation devices in the hollow fiber housing. One such
device is a screen module, consisting of a screen mash as the separation matrix. All
such separation modules will be referred to, collectively, as the filter element or
simply as the filter.
Referring to Fig. 1 there is shown a fluid filtration system according to the
invention. A process vessel or liquid storage vessel (1) is connected via a fluid
transfer line (4) to a filter containing compartment (6). The vessel (1) may be any
suitable container for a fluid to be filtered. For example, it may be a bioreactor, a
fermentor or any other vessel, nonexclusively including vats, barrels, tanks, bottles,
flasks, containers, and the like which can contain liquids. The vessel may be
composed of any suitable material such as Ultra Low Density Poly Ethylene
(ULDPE), Low Density Polyethylene (LDPE), Multilayer materials like the CX5-14
film, Polyester, Tie barrier layer, Ethyl Vinyl Alcohol (EVOH), Tie barrier layer and a
Polyester Elastomer (PE)) or a multilayer material which contains PET, PA, EVOH
and ULDPE, metal such as stainless steel, glass, or the like.
The fluid transfer line (4), also referred to as connector, or fluid connector,
serves to direct a fluid (which is to be filtered and thus may contain particulate
material, e.g. cells, but for brevity will be referred to as “fluid” herein) from the
storage vessel (1) into an entrance end (7) of the filter containing compartment (6).
The fluid transfer line (4) may comprise a vessel port, suitable for flowing fluid into
and out of the vessel (1), attached to a joint, which in turn is connected to the entrance
end (7) of the filter containing compartment (6). Suitable ports nonexclusively include
any sanitary, leak-proof fittings known in the art such as a compression, standard
Ingold or a sanitary type fitting. Suitable joints nonexclusively include pipes, tubes,
hoses, hollow joint assemblies, and the like. The joint may vary from one system to
another, based on the configuration and requirements of the vessel and process. In a
preferred embodiment, the fluid transfer line (4) is connected to the entrance end (7)
of the filter containing compartment (6) via a tube connections, such as silicone
rubber, C-flex, bioprene or dry-to-dry aseptic connections as e.g. Readymate
connections from GE Healthcare, or Kleenpack sterile connectors from Pall. The fluid
transfer line (4) may also be connected to the vessel (1) and the filter containing
compartment (6) by means of valves and suitable clamps, such as a triclamp sanitary
fitting or the like. This does not preclude the use of other appropriate connections.
In one embodiment the fluid filtration system comprises a tubing as fluid
transfer line, connected between the fluid storage vessel (1) and the filter containing
compartment (6).
In one embodiment said fluid transfer line (4) is connected at one end thereof
to the storage vessel by a valve, and attached at another end thereof to an entrance end
(7) of the filter containing compartment (6) by a valve.
The filter containing compartment (6) preferably has an entrance end (7) and
an exit end or retentate end (9). The name retentate end means that the fluid (with
possible particulate material) that is being filtered and remains within the lumen of the
filter, i.e. does not pass the pores of the filter to the other side of the membrane, which
fluid is called the retentate, can pass this end. Material that passes through the pores of
the filter is called the permeate or also the filtrate, and therefore another possible exit
end of the filter compartment, referred to as permeate port (10) or fluid harvest port
can be provided to the filter containing compartment, which permeate port allows the
tangential fluid flow to be recovered or harvested. In certain embodiments the fluid
filtration system further comprises at least one permeate pump (12) or filtrate pump
connected to the permeate port (10). The entrance end (7) of the filter containing
compartment is connected to the fluid transfer line (4). The retentate exit end (9) of
the filter containing compartment (6) is connected to an expansion chamber (17). The
filter containing compartment (6) can be connected to the expansion chamber (17)
directly by an appropriate connection, or for instance, through the intermediate of a
fluid transfer line (14) (as shown in Fig.1). In one embodiment the exit end (9) is
connected directly to the expansion chamber (17) by means of a dry-to-dry aseptic
connection, e.g. Readymate connections from GE Healthcare, and a clamp (not
shown). In another embodiment, the exit end (9) is connected to the expansion
chamber (17) by means of a fluid transfer line (14). Preferably said fluid transfer line
(14) is in the form of a tube assembly but other types of connectors are suited as well.
Said tube assembly should comprise appropriate connection means for connecting the
filter containing compartment and the expansion chamber, as e.g. dry-to-dry aseptic
connections, e.g. Readymate connections from GE Healthcare. This does not preclude
the use of other appropriate connections.
Suitable materials for the filter containing compartment (6) nonexclusively
include plastics like polysulfone, metal or glass. In preferred embodiments, materials
appropriate for gamma sterilization and preferably commonly used as disposable
materials (i.e. generally for one-time use) are suitable materials. The skilled man in
the art knows what materials are commonly used and suitable for this application.
Most preferably, the filter containing compartment is made out of disposable material,
and preferred examples include polysulfone. The filter containing compartment (6)
comprises a filter (8). Suitable filter elements nonexclusively include hollow fiber
filters, screen filters, and the like. Most preferably, the filter element is a hollow fiber
filter or filters consisting of a screen mash. Suitable hollow fiber filtration membranes
or screen filters are commonly available from various vendors, e.g. Ready to process
hollow fibers from GE Healthcare or WaterSep; Krosflo hollow fibers from Spectrum,
Microza hollow fibers from Pall. In certain preferred embodiments, the filter is
positioned longitudinally from the entrance end (7) to the exit end (9) of the filter
containing compartment (6), which enables tangential flow of the fluid along the
filter. When the filter is a hollow fiber filter, the axes of the hollow fibers preferably
extend longitudinally from the entrance end (7) to the exit end (9) of the filter
containing compartment (6).
The expansion chamber (17) is connected to the exit end (9) of the filter
containing compartment (6) on one side and to a gas flow controller (28) on the other
side. The expansion chamber (17) is capable of aspirating and receiving the fluid from
the vessel (1) through filter (8) into the expansion chamber (17) and alternately
expelling the fluid from the expansion chamber (17) back into the vessel (1) through
the filter (8). In this way, an alternating tangential flow of fluid is generated through
filter (8). Said tangential flow can be harvested through a fluid harvest port (10) into a
permeate line (11). In a preferred embodiment, said permeate line (11) comprises a
permeate pump (12) which regulates the tangential flow.
Filter (8) can be a hollow fiber filter, a mesh filter or the like. In the case
where filter (8) is a hollow fiber filter, both ends, the entrance end and the exit end of
filter (8) are sealed against the housing wall of the filter containing compartment (6)
to prevent mixing of the retentate side, and the permeate (filtered) side of the filter.
The retentate side of the fiber is the lumen side of the hollow fiber and the permeate
(or filtrate) side is the shell side of the hollow fiber. Such a leak proof seal can be
formed by a number of methods known in the art, including O rings, gaskets or any
other means that form an impenetrable barrier between the circumference at each end
of the filter and the inner wall of the housing. Alternating flow of retentate between
the expansion chamber (17) and vessel (1) is through the lumen side of the filter (8) in
the filter containing compartment (6).
The expansion chamber (17) has an entrance end, also named first opening
(16) through which fluid flows from the exit end (9) of the filter containing
compartment (6). The exit end of the expansion chamber, also named second opening
(24) is operably connected to a gas flow controller (28) by a gas line (22). In a
preferred embodiment said gas line (22) is a reversible inlet/exhaust line. In other
embodiments, separate inlet and exhaust gas lines are provided (not shown). The fluid
contained in the fluid storage vessel (1) and the filter containing compartment (6) is
alternately drawn into the expansion chamber (17) and expelled from it. The back and
forth movement of the fluid is actuated by alternately applying sufficient positive and
negative pressure into the expansion chamber. Said positive pressure, which is defined
as higher pressure than the pressure in the filter containing compartment (6) is
preferably obtained by feeding a gas, such as compressed air, through the gas line
(22). Negative pressure, which is defined as a lower pressure than the pressure in the
filter containing compartment (6), is preferably obtained by applying under-pressure
or vacuum into the expansion chamber. The positive and negative pressure can be
applied using means and methods that are well known to the skilled person, and such
means and methods need not further be elaborated here.
The expansion chamber (17) can be any type of container having any type of shape as
e.g. a cylindrical, square, or circular shape (not limiting). In certain embodiments, said
chamber has a cylindrical shape. The expansion chamber according to the present
invention contains no separation means, such as a diaphragm or any other moving
parts or physical barriers, between the first and second opening. The expansion
chamber comprises one room only and is not separated into two or more
compartments. In operations, the expansion chamber comprises a direct gas-liquid
interface, without separation means, that is formed by the liquid contained in the
system which is in direct contact with the gas phase provided by the gas flow
controller.
The chamber must be suited for containing both the liquid providing from the
vessel and the gas providing from the gas flow controller through the gas line (22).
Suitable materials for the expansion chamber nonexclusively include plastics like
polysulphon. Alternatively the chamber is made of metal such as stainless steel. In
preferred embodiments, materials that are appropriate for gamma sterilization are used
as suitable materials. In further preferred embodiments, materials that are commonly
used as single use materials are used as suitable materials. In further preferred
embodiments, materials that are appropriate for gamma sterilization and that are
commonly used as single use materials, are used as suitable materials. The skilled
man in the art would know what materials are commonly used and suitable for this
application. Single use materials are as the name states generally appropriate for a one
time use. Usually they are sterilized once, used subsequently and disposed. As
opposed to traditional materials such as e.g. stainless steel, they do not require to be
repeatedly washed, assembled and sterilized. The use of components made from
single use materials, also called disposables, in the pharmaceutical industry, offers the
advantage of not having to perform the very costly validation of the cleaning,
assembly and sterilization processes of said components. Disposable systems are
generally less expensive and a spent system can be replaced easily by an unused
system, even during the process.
The expansion chamber (17) is preferably made at least in part (e.g.
comprising a ‘window’) or substantially completely, from a transparent material in
order to visualize the liquid level in the chamber. The liquid that is drawn into the
chamber preferably should not exceed a certain maximal level within the container.
Said maximal liquid level is measured with a level sensor (24) as indicated in Fig. 1,
mounted on the surface of the container. Said level sensor (24) is named “Level
sensor high high” (LSHH). In the preferred embodiment as indicated in Fig. 1, the
container optionally indicates two levels on its surface: level sensor low (LSL) and
level sensor high (LSH), which are measured by a level sensor low (25) and a level
sensor high (26). In preferred embodiments, during the filtration process, the liquid
level should normally fluctuate approximately between said LSL and LSH. When
expelled from the expansion chamber (17), the liquid should preferably not drop
below approximately the LSL level. When aspirated from the fluid storage vessel (1)
into the expansion chamber (17), the liquid should preferably not go higher than
approximately the LSH level.
In preferred embodiments, the expansion chamber (17) is further provided
with two level sensors, preferably mounted on the expansion chamber’s surface,
which determine the fluid level in the expansion chamber (17) and provide feedback
to the gas flow controller (28), which in turn actuates the alternating positive and
negative pressure cycle in the expansion chamber. Level sensors may alternatively be
inside the expansion chamber (17), but it is preferred to mount them on its surface
(i.e. on the exterior side of the expansion chamber), since contact with the filtered
fluid (which is inside and may contact an interior side of the expansion chamber) is
then avoided. Level sensors as such are known in the art and can use a variety of
parameters to measure the level of fluid in the expansion chamber, e.g. the
K1R87xXT2 from Wenglor which is based on light scattering, or the sensor from
Aquasant Messtechnik based on capacity measurements. In a preferred embodiment,
microwave emitters, e.g. Vegamip from Vega, are used for level detection. Said
sensors are particularly accurate in the presence of foam on the medium surface.
The exit end (18) of the expansion chamber (17) is connected to a gas line
(22), which in turn is connected to a gas flow controller (28). Preferably, the gas line
(22) comprises a sterile filter (21) in order to provide for sterile gas, e.g. compressed
air, into the expansion chamber. Herewith, the risk of contaminating the liquid phase
in the expansion chamber is minimized. In a preferred embodiment said sterile filter
(21) is an air filter which preferably is provided with a heater in order to prevent
blockage of the filter due to wetting by vapor generated in the expansion chamber.
When the gas line (22) comprises a sterile filter (21), as depicted in Fig.1, the filter is
further connected to the expansion chamber (17) by means of an additional gas line
(20).
The gas flow controller (28) provides for positive and negative pressure
through the gas line (22), which preferably is a reversible inlet/exhaust line. The
positive pressure can for instance be generated directly, for instance via a compressed
air supply. The negative pressure is generated in the controller, for instance by
creating a vacuum. The vacuum can be generated by a vacuum pump or for instance a
vacuum injector. A vacuum injector is a pump-like device that uses the Venturi effect
of a converging-diverging nozzle to convert the pressure energy of a motive fluid to
velocity energy which creates a low pressure zone that draws in and entrains a suction
fluid. After passing through the throat of the injector, the mixed fluid expands and the
velocity is reduced which results in recompressing the mixed fluids by converting
velocity energy back into pressure energy. The motive fluid may be a liquid, steam or
any other gas. The entrained suction fluid may be a gas, a liquid, a slurry, or a dust-
laden gas stream. In a preferred embodiment, the gas flow controller does not require
a separate vacuum supply. Alternatively to a vacuum injector, other known means and
methods for creating under-pressure in the expansion chamber (17) may be used.
Instead of compressed air, other gases or gas mixtures may be used, e.g. nitrogen,
nitrogen/oxygen or nitrogen/oxygen/carbion dioxide mixtures, etc.
During the filtration process, the liquid contained in the fluid storage vessel (1)
is aspirated out of the vessel (1), through the filter (8), into the expansion chamber
(17) and alternately expelled from it back into the vessel (1). When the liquid is
aspirated out of the vessel (1), negative pressure is applied until the level sensor LSH
(26) is responding, that is until the liquid is drawn into the expansion chamber (17)
and the level in the chamber (17) reaches an upper limit (LSH). This triggers a switch
to apply positive pressure. The positive pressure is then applied until the level detector
LSL (25) is responding, that is until the liquid is expelled from the expansion chamber
(17) and the liquid level in the chamber (17) reaches a lower limit (LSL). This triggers
again the switch to apply negative pressure. As a result, the fluid in the system flows
back and forth through the filter (8) in a controlled way, providing for a tangential
fluid flow into the permeate line (11).
Gas flow controllers as such are known and can be used according to methods
as such known by the skilled person, in accordance with the present disclosure. The
gas flow controller (28) provides for positive and negative pressure into the expansion
chamber (17). In certain embodiments, the gas flow controller (28) may comprise a
pressure measuring device (32), such as a pressure sensor which serves to monitor
and/or regulate the pressure in the gas line (22). In addition, the gas flow controller
may comprise a pressure measuring device (30) which serves to measure the pressure
in the permeate line (11). In certain embodiments, the gas flow controller (28) is
connected to an air or other gas supply, which provides the gas flow controller with
air or gas, from which the pressure can optionally be reduced with a pressure reducer
(46). The gas which may be reduced in flow is further directed either through a
pressure controller (44) and control valve (40) towards the gas line (22) in order to
provide for the positive pressure; or alternatively through a pressure controller (42)
and vacuum injector (36) in order to provide for negative pressure into the gas line
(22) and the expansion chamber (17). In addition, the gas flow controller may
comprise a shut off valve (38), which is functionally in contact with the level sensors
(25, 26) and which closes when the fluid in the expansion chamber (17) has reached
the maximal level (LSH). The gas flow controller (28) further comprises a switch over
valve (34), which is in contact with the level sensors (25, 26) and which determines
whether compressed air (having higher pressure than the pressure in the filter
containing compartment) or vacuum or under-pressure (as compared to the pressure in
the filter containing compartment) is applied into the gas line (22).
In one embodiment, the liquid level in the expansion chamber is controlled by
level sensors, such as light switches or microwave emitters. Several of said level
sensors are positioned along the expansion chamber and allow to calculate the speed
of the medium front in the expansion chamber. The speed of the medium can be
determined by continuously measuring the time (∆t) spent by the medium between the
LSL level and LSH level. Variations in speed can indicate that the hollow fiber
membrane is about to clog or that it has clogged. By measuring the speed, it is
possible to automatically control the positive and negative pressure cycle and
therewith adjust the medium flow rate or adjust the medium aspiration / expulsion
cycle, in order to unclog the hollow fiber that shows signs of clogging.
In another embodiment, the positive and negative pressure cycle in the
expansion chamber can be regulated based on particle size distribution and load of the
cell suspension. Particle size distribution and load can be measured with a particle
sizer, e.g. a Casy® counter. The particle size distribution and load output values can
be directed to the gas flow controller in order to regulate the positive and negative
pressure cycle. This embodiment allows the possibility to prepare a calibration curve
correlating the hollow fiber performance to the cell density and allowing automatic
flow control according to the cell density input.
This has a significant advantage over the filtration systems currently used in
which action can only be taken manually by an operator once the hollow fiber shows
signs of clogging. Hence the system according to the present invention works
automatically and pro-actively.
All these control features are not available in the currently existing filtration
systems (such as the ATF system currently commercially available and disclosed in
US 6,544,424). The cycle number is the only parameter that is controlled in said
systems. The cycle time is adapted manually by an operator based on gut-feeling.
The filter containing compartment (6) is further provided with a permeate port
(10), which allows the tangential fluid flow to be recovered. The permeate exits the
filter containing compartment through the permeate port (10) into a permeate line
(11). In the most preferred embodiment, a permeate pump (12) is connected to the
permeate line (11). The permeate pump (12) is suitable as a means for controlling the
removal of filtered fluid (permeate) from the system and to serve as a check valve to
regulate the unrestricted flow of permeate from compartment (6). Pressure in the
permeate line may be monitored by a pressure sensor (30), as shown in Fig.1.
While a single embodiment of the invention has been disclosed in detail, many
other variations are contemplated. For instance, in another embodiment of the
invention a screen mesh filter module could be used instead of the hollow fiber filter
module described above. A screen mesh that is appropriate in the present invention
was described in e.g. US 6,544,424.
In some applications it may be desirable to penetrate the process vessel (1)
through an opening other than the bottom side opening described above. Not shown is
a top penetrating port through a head plate of the process vessel (1). In this case the
relationship between system components including the fluid transfer line (4), the filter
containing compartment (6), the expansion chamber (17) and the gas flow controller
(28) remains the same. Note, however, that to generate alternating tangential flow, a
dip tube (not shown) would then be used to connect the filtration system to the liquid
in the process vessel (1). Filtered harvest (permeate) would similarly be collected
from the filter containing compartment (6), through a permeate port (10) into the
permeate line (11). The liquid volume that is removed from the system (through the
permeate line) may be restored into the system by a level control mechanism that
activates an addition pump in order to pump liquid into the vessel.
Other variations of the invention are also possible. For example, multiple filter
containing compartments may be connected in parallel to a single vessel. Each filter
containing compartment may be connected to a separate expansion chamber. When
multiple filter containing compartments are disposed in parallel formation and a first
filter is blocked, the process can continue with a second or third filter containing
compartment. Herewith the continuity of the process is guaranteed.
One further aspect of the present invention relates to an assembly as depicted
in Fig.2, comprising an expansion chamber (17) assembled to a filter containing
compartment (6) on one side and to an air filter (21) on the other side, wherein the
assembled components are preferably made of disposable materials, and preferably
are sterile (at least on the inside that is intended to be in contact with the fluid that is
to be filtered). Single use materials (or disposables) are defined previously and the
skilled person would know which materials are appropriate for this purpose.
The expansion chamber (17) of said assembly has an entrance end, also named
first opening (16) which is coupled to the exit end or retentate end (9) of the filter
containing compartment (6). Both components are connected directly to each other or
via an intermediate tube assembly (14), as shown in Fig.2. When directly connected to
each other, an appropriate connection is e.g. a dry-to-dry aseptic connection, as e.g. a
Readymate connection from GE Healthcare or a Kleenpack connection from Pall.
When an intermediate tube assembly is used, said tube assembly preferably comprises
a tubing of silicone, bioprene, C-flex or the like. Each tube end comprises a
connecting means in order to appropriately connect the intermediate tube assembly
(14) to the filter containing compartment (6) and the expansion chamber (17). Suited
connections can for instance be dry-to-dry aseptic connection, as e.g. Readymate
connections or the like.
The exit end, also named second opening (18) of the expansion chamber is
connected to an air filter (21). Both components are connected directly to each other
or via an intermediate tube assembly (20), as shown in Fig.2. Appropriate connections
are similar to those connecting the expansion chamber to the filter containing
compartment.
In one embodiment of the present invention, the expansion chamber of the
assembly contains no separation means, such as a diaphragm or any other moving
parts or physical barriers, between the first and second opening. The expansion
chamber comprises one room only and is not separated into two or more
compartments. Herewith, the expansion chamber, when built in said assembly and
when connected to a gas flow meter and a fluid storage vessel according to the
invention, comprises a direct gas-liquid interface without separation means that is
formed by the liquid contained in the system which is in direct contact with the gas
phase provided by the gas flow controller.
In one embodiment, the assembly will be sterilized, preferably by gamma
irradiation, and packaged for storage. The pre-sterilized assembly can be connected
sterilely to a fluid vessel and a gas flow controller in order to perform tangential
filtration according to the invention. The advantage of said assembly is that it contains
no moving parts and is therefore not prone to wastage or breakage of the system
during the filtration process. In a second place the assembly, which is pre-sterilized,
allows the filtration process to start right after it has been connected. No costly
cleaning and sterilization validation procedures are required.
Another aspect of the present invention relates to a method for preparing an
assembly as previously described comprising the steps of assembling a filter
containing compartment on to a first opening of an expansion chamber and
assembling an air filter on to a second opening of said expansion chamber.
Another aspect of the present invention relates to a kit of parts comprising a
filter containing compartment, an expansion chamber and a sterile air filter, which can
be used for providing an assembly as described herein (Fig. 2). The components of the
kit of parts are preferably disposables. The kit of parts provides for a very robust and
cost effective assembly to be used in a system for tangential flow filtration according
to the invention.
The system of the present invention may be used for filtering, concentrating,
clarifying or otherwise conditioning a fluid or process medium when used with the
appropriate filter element. The system may also be used as a sampler, suitable for
extracting filtered stream from a process vessel while allowing continuous monitoring
and analysis of the process. The back and fourth flow of medium between vessel and
filter allows continuous equilibration of the process medium between these two
compartments. In certain embodiments, the filtered stream may therefore be
considered representative of the content in the vessel.
The system of the present invention is most preferably used for filtering fluids,
most preferably biological fluids. Nonexclusive examples of biological fluids include
suspension cultures, microcarrier based cultures, blood, and other fluids containing
animal, microbial or plant cells. Although a preferred embodiment of the present
invention is disclosed with regard to filtering a biological fluid, it is understood that
the present invention can be used for filtering other liquids.
In a preferred embodiment, the system of the present invention can be used for
the production of biologicals such as recombinant proteins as described in e.g. WO
2008/006494, or such as viruses as described in . The filtration
system used in said references could be replaced by the PACS system.
Example 1: Use of PACS system for culturing cells in perfusion
PER.C6 cells were precultured in Permexcis® medium in a Cultibag rocking
motion system. Subsequently said cells were transferred for inoculation into a stirred
10L bioreactor which was connected to a PACS system according to the present
invention. Permeate withdrawal using the PACS system was started when a cell
density of approximately 2x10 viable cells/mL was reached (2 days post inoculation).
After 9 days of perfusion, the cell culture reached a cell density of more than 60x10
viable cells/mL, as shown in Fig. 3. This experiment shows that the PACS system can
be successfully used for growing cells in perfusion.
Example 2: Use of PACS system for culturing cells at high cell densities and
propagating adenovirus.
PER.C6 cells were thawed and propagated in serum free culture medium in a
20L Wave bioreactor® at a working volume of 10L. The cells were propagated at
37°C with a CO and air overlay of 0.18 L/min and 20 mL/min respectively. Cell
counts were performed on a daily basis. On day 4 after inoculation of the Wave
bioreactor® a back dilution was performed. Day 7 post inoculation the cell suspension
was used to inoculate a 10L stirred bioreactor, coupled to a PACS system, at a cell
density of 0.57x10 total cells/mL. Cells were propagated in the 10L bioreactor at
37°C, DO of 40%, and a pH of 7.3. The PACS system was started directly after
inoculation of the bioreactor, perfusion was started 2 days post inoculation at a cell
density of approximately 2x10 viable cells/mL. After 8 days of perfusion, a cell
density of 83.2x10 viable cells/mL was reached. The cell growth is presented in Fig.
On day 10 post inoculation, the suspension contained in the 10L bioreactor
was transferred to a 50L disposable bioreactor containing 40L fresh medium. This
resulted in a cell density of 20.94x10 total cells/mL (90% viability, hence 18.86x10
viable cells/mL) at inoculation.
Subsequently the 50L bioreactor, which was connected to a PACS system, was
infected with an Ad35 adenovirus at an MOI of 70 virus particles/cell (VP/cell) and
incubated at 36°C, pH 7.3 and DO of 40%. The PACS system was started 5 hours
post infection at a medium refreshment rate of 2 vessel volumes per day for the
subsequent 40 hours. On day 3 post infection the 50L bioreactor was sampled for
measuring virus production by AEX-HPLC. To release the virus from the cells 1 mL
sample was mixed with 100 µL 10% Triton X-100 and incubated at 37°C for 30
minutes. After incubation the sample was mixed with 2.42 µL benzonase/MgCl
followed by a subsequent incubation step of 30 minutes at 37°C. Finally 100 µL 50%
sucrose was added to the samples. After a centrifugation step of 5 minutes at 2500g
the samples were stored at a temperature below -65°C until analysis by AEX-HPLC.
The AEX-HPLC results showed that a yield of 2.08x10 VP/mL was reached on day
3 post infection.
These results demonstrate that the PACS system can be used for culturing
cells at very high cell densities, up to more then 80x10 viable cells/mL. Furthermore,
these results show that the PACS system can be used for propagating virus on high
cell density cultures and increasing the volumetric yields almost 10 times compared to
a batch process.
Claims (28)
1. A fluid filtration system comprising: a) at least one fluid storage vessel; 5 b) at least one filter containing compartment; c) at least one fluid transfer line connecting the storage vessel to an entrance end of a filter containing compartment, which fluid transfer line is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment; d) at least one expansion chamber with at least two openings, wherein a first opening 10 is connected at an exit end of the filter containing compartment and wherein a second opening is connected to a gas flow controller; wherein the expansion chamber contains no separation means between the first and second opening; and wherein at least one level sensor is mounted on or in the expansion chamber; e) at least one gas flow controller for alternately providing the expansion chamber 15 with positive and negative pressure; f) at least one fluid harvest port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment.
2. A fluid filtration system according to claim 1, wherein the expansion chamber 20 is connected to the gas flow controller with a gas line.
3. A fluid filtration system according to any one of claims 1-2, wherein the gas line comprises a filter between the expansion chamber and the gas flow controller.
4. A fluid filtration system according to claim 3, wherein the filter is a sterile 25 filter.
5. A fluid filtration system according to claim 3, wherein the filter comprises a sterile filter with heater. 30
6. A fluid filtration system according to any one of claims 1-5, wherein the fluid storage vessel is a bioreactor.
7. A fluid filtration system according to any one of claims 1- 6, wherein the filter containing compartment contains a hollow fiber filter.
8. A fluid filtration system according to any one of claims 1-7, wherein at least two level sensors are mounted on or in the expansion chamber. 5
9. A fluid filtration system according to any one of claims 1-8, wherein the level sensor is or level sensors are mounted on the surface of the expansion chamber.
10. A fluid filtration system according to any one of claims 1-9, wherein said level sensors are capable of measuring a minimal and a maximal fluid level in the 10 expansion chamber, and functionally coupled to the gas flow controller.
11. A fluid filtration system according to any one of claims 1-10, capable of alternately aspirating the fluid of the system from the storage vessel, through the filter containing compartment into the expansion chamber, and expelling the fluid from said 15 expansion chamber through the filter containing compartment back into the storage vessel, wherein fluid aspiration is performed by applying negative pressure into the expansion chamber and fluid expulsion is performed by applying positive pressure into the expansion chamber. 20
12. A fluid filtration system according to any one of claims 1-11, wherein said negative pressure is obtained by creating a vacuum in the expansion chamber and positive pressure is obtained by injecting gas into the expansion chamber.
13. A fluid filtration system according to claim 12, wherein the gas is compressed 25 air.
14. A fluid filtration system according to any one of claims 1- 13, wherein the filter in the filter containing compartment comprises a plurality of bundled hollow fibers whose axes extend longitudinally from the entrance end to the exit end of the 30 filter containing compartment.
15. A fluid filtration system according to any one of claims 1-14, wherein the filter containing compartment and the expansion chamber are disposable.
16. A fluid filtration system according to claim 15, the fluid storage vessel and the fluid transfer line are disposable.
17. A process for filtering a fluid comprising: 5 a) providing a fluid filtration system comprising at least one fluid storage vessel; at least one filter containing compartment; a fluid transfer line connecting the storage vessel to an entrance end of the filter containing compartment, which transfer line is capable of directing a fluid from the storage vessel into the entrance end of the filter containing compartment; 10 at least one expansion chamber connected on one side to an exit end of the filter containing compartment allowing for liquid from said compartment to enter the chamber and on the other side to a gas flow controller, which alternately provides negative and positive pressure into the expansion chamber thereby aspirating fluid from the exit end of the filter containing compartment and expelling fluid back into 15 the exit end of the filter containing compartment, forming a direct gas-liquid interface without separation means into the expansion chamber; and at least one permeate port connected to the filter containing compartment for removing filtered fluid from the filter containing compartment; b) drawing fluid out of the storage vessel through the filter into the expansion 20 chamber by applying negative pressure into the expansion chamber; c) expelling the fluid from the expansion chamber through the filter back into the storage vessel by applying positive pressure into the expansion chamber; d) repeating steps b and c generating an alternative tangential flow of fluid through the filter; and 25 e) removing the filtered fluid from the filtration system.
18. A process for filtering a fluid according to claim 17, wherein the fluid storage vessel is a bioreactor. 30
19. A process for filtering a fluid according to any one of claims 17-18, wherein the filter containing compartment and/or the expansion chamber are disposable.
20. A process for filtering a fluid according to claim 19 wherein the filter containing compartment, the expansion chamber, the fluid storage vessel and the fluid transfer line are disposable. 5
21. A process for filtering a fluid according to any one of claims 17-20, wherein the positive and negative pressure are regulated by level sensors which measure the fluid level in the expansion chamber.
22. A process for filtering a fluid according to claim 21, wherein the level sensors 10 are mounted on the expansion chamber.
23. A process for filtering a fluid according to any one of claims 17-22, wherein the filter containing compartment contains a hollow fiber filter. 15
24. A process for filtering a fluid according to any one of claims 17-23, wherein said negative pressure is obtained by creating a vacuum in the expansion chamber and positive pressure is obtained by injecting gas into the expansion chamber.
25. A process for filtering a fluid according to claim 24, wherein the gas is 20 compressed air.
26. A fluid filtration system according to claim 1, substantially as herein described with reference to any embodiment disclosed. 25
27. A fluid filtration system substantially as herein described with reference to any embodiment shown in the accompanying drawings.
28. A process for filtering a fluid according to claim 17, substantially as herein described with reference to any embodiment disclosed.
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161463095P | 2011-02-10 | 2011-02-10 | |
US61/463,095 | 2011-02-10 | ||
EP11153995.3 | 2011-02-10 | ||
EP11153995 | 2011-02-10 | ||
EP11183676.3 | 2011-10-03 | ||
EP11183676 | 2011-10-03 | ||
US201161627812P | 2011-10-18 | 2011-10-18 | |
US61/627,812 | 2011-10-18 | ||
PCT/EP2012/052031 WO2012107436A1 (en) | 2011-02-10 | 2012-02-07 | Pneumatic alternating pressure membrane cell separation system |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ614364A NZ614364A (en) | 2015-07-31 |
NZ614364B2 true NZ614364B2 (en) | 2015-11-03 |
Family
ID=
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