A continuous fermenter vessel such as an air-lift fermenter for
receiving and cultivating a suspension culture of cells. The vessel
contains a mechanism for agitating the suspension culture received
in the vessel and causing a gross flow movement of the suspension
culture. The vessel includes an inlet for continuously supplying
culture medium to the vessel and an outlet for removing the culture
supernatant. A filter material is disposed inside the vessel so
that the gross flow movement of the suspension culture substantially
prevents clogging of the filter material by cells and/or cell debris.
1. A continuous air-lift fermenter vessel for receiving a suspension
culture of cells, the vessel comprising:
air-lift means, in use for agitating a suspension culture received
in the vessel and causing a gross flow movement of the suspension
culture, an inlet for continuously supplying culture medium to the
vessel, an outlet for removing culture supernatant, and a filter
material between the outlet and the vessel to prevent removal of
cells from the suspension culture wherein the filter material is
located such that in use, the gross flow movement of the suspension
culture inhibits clogging of the filter material by cells and/or
cell debris, said the outlet comprising a conduit formed in a divider
between a downcomer and a riser, the conduit communicating with
the fermenter vessel via the filter material forming at least a
part of the divider, the divider comprising a double-walled draught
tube defining, between the double-walls, a conduit space communicating
in use with the suspension culture through at least a portion of
the draught tube comprising the filter material.
2. A continuous fermenter vessel according to claim 1 wherein the
filter material has a pore size of from 1 to 10 .mu.m.
3. A continuous fermenter vessel according to claim 1 wherein the
inside of the draught tube is formed of an impermeable material
and at least a portion of the outside of the draught tube is formed
of the filter material.
4. A method for the suspension culture of cells comprising culturing
the cells in a continuous fermenter vessel according to claim 1.
5. A method according to claim 4 wherein the cells are hybridoma
6. A continuous air-lift fermenter vessel for receiving a suspension
culture of cells, said vessel comprising:
an inlet means for continuously supplying culture medium to said
a divider comprising a filter material and a double-walled draught
tube having two concentric walls and a conduit space therebetween,
said conduit space communicating with said vessel via said filter
a riser and a downcomer each having an upper and lower part and
being separated from each other by said divider;
means for injecting a gas into said lower part of said riser for
causing a gross flow movement of suspension culture in said vessel
comprising an upward flow of suspension culture in said riser and
a downward flow of suspension in said downcomer, said gross flow
movement of suspension culture allowing culture supernatant to be
drawn into said conduit space and inhibiting clogging of said filter
material with cells from said suspension culture; and
outlet means for removing culture supernatant from said conduit
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a continuous fermenter vessel for receiving
an agitated suspension culture of cells.
2. Description of the Prior Art
Recent advances in the commercialization of products produced by
the in vitro fermentation of cells have led to a growing interest
in the design of improved fermenter vessels and fermentation processes.
Fermentation is usually carried out either in a batch process or
in a continuous process. Continuous processes are advantageous since
they enhance the productivity of a given fermenter and reduce the
downtime necessary for cleaning and sterilization which is normally
required in a batch fermenter.
In known continuous fermenter vessels, a suspension of cells in
an appropriate culture medium is agitated and maintained at a suitable
temperature for fermentation. Suspension culture is continuously
withdrawn from the fermenter, balanced by a continuous supply of
fresh culture medium. A significant disadvantage of such fermenters
is the continual loss of cells caused by the removal of culture.
Fermenters are known in which the removed suspension culture is
passed through a continuous centrifugal separation device which
separates cells from the culture supernatant. The cells are then
fed back into the suspension culture.
In another known fermenter, a rotating basket of a filter material
is provided, partially submerged in the culture, such that the inside
of the basket is separated from the suspension culture by the filter.
Culture supernatant passes through the filter and may be withdrawn
continuously, whilst cells remain in the suspension culture. The
rotation of the basket in the suspension culture reduces clogging
of the filter material.
These known devices for providing cell feedback in continuous suspension
cultures are complicated mechanically, require energy for their
operation and may cause detrimental effects, such as cell rupture,
upon the suspension culture. These features combine to reduce the
economic viability of fermentation processes based on such fermenters.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a continuous
fermenter vessel which substantially overcomes these disadvantages.
According to the present invention there is provided a continuous
fermenter vessel for receiving a suspension culture of cells, the
vessel comprising; means, in use, for agitating a suspension culture
received in the vessel and causing a gross flow movement of the
suspension culture, an inlet for continuously supplying culture
medium to the vessel, an outlet for removing culture supernatant,
and a filter material between the outlet and the vessel to prevent
removal of cells from the suspension culture wherein the filter
material is located such that, in use, the gross flow movement of
the suspension culture substantially prevents clogging of the filter
material by cells and/or cell debris.
The fermenter vessel of the invention requires no moving parts
in order to achieve separation of the culture supernatant from the
suspension and therefore provides an advantageous saving in costs.
The filter material has a pore size which depends upon the diameter
of the cells to be cultured. For animal cells the filter material
may have a pore size of from 1 to 10 .mu.m, 1 to 5 .mu.m, and most
preferably 3 to 5 .mu.m. The filter material may be a ceramics material,
sintered stainless steel or a stainless steel mesh.
Gross flow movement of the culture supernatant may be produced
using a pump or impeller. However such devices add to the cost and
complexity of the fermenter. In a preferred form of the invention
the fermenter is a so called "air-lift" fermenter in which
a gas such as air is injected into an upwardly extending part of
the fermenter known in the art as a "riser". The riser
communicates at top and bottom with the top and bottom respectively
of a further upwardly extending part of the fermenter known in the
art as a "downcomer". A known configuration of an air-lift
fermenter comprises a central divider in the fermenter vessel separating
the vessel into two parts (riser and downcomer). An alternative
configuration of air-lift fermenter comprises a draught tube substantially
concentric with a cylindrical fermenter vessel, dividing the fermenter
into a riser (within the draught tube) and a downcomer (in the annular
space between the draught tube and the inside of the fermenter vessel).
(The riser could equally be the annular space between the draught
tube and the inside of the fermenter vessel, and the downcomer could
be within the draught tube). The injection of a gas, such as air,
into the lower part of the riser causes a reduction in the bulk
density within the riser resulting in an upward flow of liquid in
the riser, thus displacing the contents of the downcomer which circulate
back into the bottom of the riser. In this way a recycling fluid
flow is caused, mixing the culture and maintaining the cell suspension.
The advantages of such a fermenter are that no moving parts are
necessary and oxygenation of the culture occurs. Typically the cross-sectional
area of the riser is substantially the same as the cross-sectional
area of the downcomer.
In a preferred aspect of the invention, the continuous fermenter
vessel is an air-lift fermenter and the outlet comprises a conduit
formed in a divider between a downcomer and a riser, the conduit
communicating with the fermenter vessel via a filter material forming
at least a part of the divider. In this way, suspension culture
either flowing up the riser or down the downcomer flows tangentially
across the filter material substantially preventing clogging of
the filter with cells from the culture. Culture supernatant may
be drawn into the conduit within the divider and removed from the
conduit via an outlet port. Preferably the divider comprises a double-walled
draught tube defining, between the double-walls, a conduit space
communicating in use with the suspension culture through at least
a portion of the draught tube comprising a filter material. The
draught tube may be formed such that the inside of the draught tube
(i.e. in the riser) is formed of a continuous material and the outside
of the draught tube (i.e. in the downcomer) is formed of a filter
material such as sintered or mesh stainless steel.
The circulation flow rate may be increased by increasing the gas
flow rate and hence the circulation velocity. To prevent over oxygenation
of the fermenter contents, the air may be mixed with an inert gas,
such as nitrogen, in order to maintain a sufficiently high overall
gas flow rate while limiting the oxygen transfer rate. An increase
in the aspect ratio (height:diameter) of the fermenter will cause
an increase in the circulation distance and hence for a specific
circulation flow rate (which is generated by specific air flow rate)
the circulation velocity will be increased.
The fermenter may be used for the culture of any cells capable
of in vitro growth in suspension liquid culture (including microcarrier
culture), but is especially useful for the culture of animal cells
such as, for example, hybridoma cell lines.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is now illustrated by the following description with
reference to the accompanying drawing, which is a schematic axial
cross-section of an air-lift fermenter of the invention including
a double-walled draught tube concentric with a fermenter vessel.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1 an air-lift fermenter comprises a cylindrical
outer vessel shown generally at 1. A draught tube shown generally
at 3, is located concentrically within the outer vessel 1. The draught
tube consists of a double-walled cylinder comprising inner and outer
cylindrical walls, 5, 7 respectively, sealed at each end by annular
caps 11 and 13 forming an annular conduit 9. At least a portion
of the inner and/or outer walls 5 and 7 of the cylindrical draught
tube 3 comprises a filter material capable of allowing passage of
culture supernatant but preventing the passage of cells. A suitable
filter material is sintered stainless steel or a stainless steel
mesh. The pore size of the filter depends upon the cells to be cultured.
In the case of animal cells a pore size in the range 1 to 10 .mu.m
is effective. The inside of the draught tube 3, in use, acts as
the riser 15 and the annular space between the outer wall 7 of the
draught tube 3 and the inner wall of the outer vessel 1 acts as
the downcomer 17.
The base of the outer vessel 1 carries an air inlet 19 directly
below and approximately central with respect to the riser 15. The
top of the conduit 9 is provided with an outlet 21 for withdrawing
culture supernatant. The fermenter is provided with an inlet 23
for supplying culture medium to the fermenter. Thermostatically
controlled heating means may be provided either in or around the
outer vessel 1. The outer vessel 1 may be double-walled to provide
a jacket, for example filled with water.
In use, a suspension culture of cells, suitably animal cells, is
introduced into the fermenter such that the top of the draught tube
3 is covered by a depth of the suspension culture corresponding
to from 0.25 to 1.0 times the diameter of the outer vessel 1. The
suspension culture is maintained in a flow condition by forcing
air through air inlet 19. The air rises within the riser 15 reducing
the bulk density of the liquid suspension in the riser 15 and causing
a gross flow movement of liquid in the direction indicated by the
arrows in FIG. 1. At the top of the riser 15, air within the suspension
culture is disengaged. A continuous supply of culture medium is
provided through inlet 23. The culture medium includes nutrients
and other factors necessary for the efficient culture of the cells.
Culture supernatant is continuously drawn through the filter material
forming at least a part of the double-walls 5 and 7 of the draught
tube 3. Culture supernatant drawn into the conduit 9 is then withdrawn
from the fermenter through outlet 21. The filter material may form
a part or all of either or both of the double-walls 5 and 7 of the
draught tube 3.
Experiments have revealed that for a range of fermenter sizes of
100 liters to 1,000 liters with aspect ratios (height:diameter)
of from 8 to 12 and using an air flow rate of 0.1 vvm, the circulation
velocity in both the riser and downcomer ranges from 0.15 ms.sup.-1
to 0.25 ms.sup.-1. The geometry of the riser and downcomer and the
air flow rate may be adjusted in order to achieve a sufficient circulation
velocity to prevent clogging of the filter. The pressure of the
air in the headspace may be increased to achieve a greater flow
through the filter if clogging does occur.
A continuous culture was run using a 301 continuous air-lift fermenter
provided with a nominal 10 .mu.m pore size stainless steel mesh
filter. The fermenter was substantially as shown in the accompanying
FIGURE wherein the outer wall 7 comprises a nominal 10 .mu.m stainless
steel mesh ("Hollander Twill Weave"--Simon Cadish &
Sons, U.K.) on a much larger hole size backing cylinder. The inner
wall 5 comprises a continuous stainless steel cylinder. The draught
tube is sealed such that any liquid pumped from the vessel must
have entered the space between the inner 5 and outer 7 walls through
the filter material.
A hybridoma cell-line was cultured in the vessel using a culture
medium comprising DMEM, 3.7 g/l NaHCO.sub.3 buffer and 5% foetal
calf serum, at a dilution rate of approximately 0.03 hr.sup.-1.
The viable cells in the culture and withdrawn from within the draught
tube were counted. This indicated that only about 5% of the total
viable cells in the culture were passing through the filter.
It will be understood that the present invention has been described
above purely by way of example, and modifications of detail can
be made within the scope and spirit of the invention.