U.S. patent application number 13/148503 was filed with the patent office on 2011-12-22 for bioreactor for the cultivation of mammalian cells.
Invention is credited to Mohsan Khan.
Application Number | 20110312087 13/148503 |
Document ID | / |
Family ID | 40848586 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110312087 |
Kind Code |
A1 |
Khan; Mohsan |
December 22, 2011 |
BIOREACTOR FOR THE CULTIVATION OF MAMMALIAN CELLS
Abstract
Large-scale bioreactors having at least two impellers,
large-scale bioreactor systems and methods for the large-scale
cultivation and propagation of mammalian cells using these
bioreactors.
Inventors: |
Khan; Mohsan;
(Hertfordshire, GB) |
Family ID: |
40848586 |
Appl. No.: |
13/148503 |
Filed: |
February 9, 2010 |
PCT Filed: |
February 9, 2010 |
PCT NO: |
PCT/EP2010/000783 |
371 Date: |
August 23, 2011 |
Current U.S.
Class: |
435/325 ;
435/289.1; 435/294.1 |
Current CPC
Class: |
C12M 27/20 20130101;
C12N 2527/00 20130101; C12M 21/08 20130101; C12M 27/02 20130101;
C12N 5/00 20130101; C12M 27/08 20130101; C12M 23/58 20130101; C12M
29/06 20130101 |
Class at
Publication: |
435/325 ;
435/289.1; 435/294.1 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12N 5/071 20100101 C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2009 |
EP |
09001755.9 |
Claims
1. A bioreactor for the cultivation of mammalian cells wherein said
bioreactor has a volume of at least 4000 l and at least one top
impeller and at least one bottom impeller, wherein the top impeller
is a hydrofoil impeller.
2. The bioreactor according to claim 1, wherein the top impeller is
a three bladed hydrofoil design impeller, and the bottom impeller
is a four pitched-bladed high solidity impeller.
3. The bioreactor according to claim 1, wherein the impeller to
tank diameter ratio is at least 0.35 and at most 0.55.
4. The bioreactor according to claim 1, wherein a top impeller
power number (N.sub.p) is at least 0.1 and at most 0.9.
5. The bioreactor according claim 1, wherein a top impeller flow
number (N.sub.q) is at least 0.4 and at most 0.9.
6. The bioreactor according to claim 1, wherein a bottom impeller
power number (N.sub.p) is at least 0.5 and at most 0.9.
7. The bioreactor according to claim 1, wherein a bottom impeller
flow number (N.sub.q) is at least 0.50 and at most 0.85.
8. The bioreactor according to claim 1, wherein the bioreactor has
at least one sparger.
9. The bioreactor according to claim 1, wherein the bioreactor has
at least one baffle.
10. The bioreactor according to claim 1, wherein the bioreactor has
at least two ports for alkali addition.
11. The bioreactor according to claim 1, wherein the bioreactor has
a volume of at least 10000 l.
12. A method for cultivating and propagating mammalian cells,
wherein at least one mammalian cell is cultivated under suitable
conditions and in a suitable culture medium in a bioreactor
according to claim 1.
13. The method according to claim 12, wherein an agitation rate of
the at least two impellers is at least 55 W/m.sup.3 and at most 85
W/m.sup.3.
14. A bioreactor system for the cultivation of mammalian cells
wherein a) a first bioreactor with a volume of at least 500 l is
connected with b) a second bioreactor with a volume of at least
2000 l, wherein the second bioreactor has a volume greater than the
first bioreactor and wherein the second bioreactor is connected
with c) a third bioreactor according to claim 1 having a volume of
at least 10 000 l, wherein the third bioreactor has a volume
greater than the second bioreactor.
15. The bioreactor system according to claim 14, wherein at least
one of the first or second bioreactors is a bioreactor for the
cultivation of mammalian cells, said bioreactor having a volume of
at least 4000 l and at least one top impeller and at least one
bottom impeller, wherein the top impeller is a hydrofoil
impeller.
16. A method for cultivating and propagating mammalian cells,
wherein a) at least one mammalian cell is cultivated under suitable
conditions and in a suitable culture medium in a first bioreactor
with a volume of at least 500 l, b) the medium containing the cells
obtained by propagation from the at least one mammalian cell is
transferred into a second bioreactor with a volume of at least 2000
l, c) the transferred cells are cultivated in the second
bioreactor, d) the medium containing the cells obtained in step c)
is transferred into a third bioreactor with a volume of at least
10000 l, and e) the transferred cells are cultivated in the third
bioreactor.
17. The method according to claim 16, wherein at least one of the
first, second and third bioreactors is a bioreactor for the
cultivation of mammalian cells, said bioreactor having a volume of
at least 4000 l and at least one top impeller and at least one
bottom impeller, wherein the top impeller is a hydrofoil
impeller.
18. The method according to claim 16, wherein the
cultivation-conditions are the same in the bioreactors of steps a),
c) and e).
19. The bioreactor according to claim 3, wherein the impeller to
tank diameter ratio is at least 0.40 and at most 0.48.
20. The bioreactor according to claim 3, wherein the impeller to
tank diameter ratio is at least 0.44 and at most 0.46.
21. The bioreactor according to claim 10, wherein said at least two
ports are spatially separated from one another.
22. The bioreactor according to claim 11 has a volume of at least
20000 l.
23. The bioreactor system according to claim 14, wherein the first
bioreactor has a volume of at least 1000 l.
24. The bioreactor system according to claim 14, wherein the second
bioreactor has a volume of at least 4000 l.
25. The bioreactor system according to claim 14, wherein the third
bioreactor has a volume of at least 20000 l.
26. The method according to claim 16, wherein the first bioreactor
has a volume of at least 1000 l.
27. The method according to claim 16, wherein the second bioreactor
has a volume of at least 4000 l.
28. The method according to claim 16, wherein the third bioreactor
has a volume of at least 20000 l.
Description
[0001] The present invention relates to bioreactors and methods for
the large scale cultivation of mammalian cells using these
bioreactors.
[0002] It is important in mammalian cell culture processes to
maintain the physicochemical environment in view of dissolved
oxygen, culture pH, temperature and shear sensitivity. Also the
maintenance of the nutritional environment is important. The
maintenance of the cultivation conditions limits the possibility to
perform large scale culturing of mammalian cells. Especially
concentration gradients can inhibit the cell growth of mammalian
cells in large-scale bioreactors.
[0003] One of the objects of the present invention is to provide
bioreactors and methods, which allow the cultivation of mammalian
cells in large scale volumes. Furthermore, it is an object of the
present invention to provide bioreactors and methods, which allow
the cultivation of mammalian cells under optimal conditions, even
if grown in large scale volumes and therefore allow a process
performance and product quality independent of the size of the
bioreactor.
[0004] It is an object of the present invention to provide
large-scale bioreactors which allow the cultivation of mammalian
cells in a homogenous environment with respect to process
parameters such as pH, dissolved oxygen tension (DOT) and
temperature, maintaining a well mixed cell suspension and blending
nutrient feeds within the bioreactor.
[0005] Furthermore it is an object of the present invention to
provide devices and methods which allow the production of mammalian
cells and products of the mammalian cells, especially proteins,
peptides, antibiotics or amino acids, synthesised by the mammalian
cells, in a large-scale manner.
[0006] The present invention solves the technical problems
underlying the present invention by the provision of bioreactors,
bioreactor systems and methods for the cultivation of eukaryotic
cells, especially of mammalian cells, according to the claims.
[0007] The present invention solves the technical problem
underlying the present invention especially by the provision of a
bioreactor for the cultivation of mammalian cells, characterised in
that said bioreactor has at least two impellers. Furthermore, the
present invention solves the technical problem underlying the
present invention by the provision of a method for the cultivation
and propagation of mammalian cells characterised in that at least
one mammalian cell is cultivated under suitable conditions and in a
suitable culture medium in a bioreactor, which has at least two
impellers. Furthermore, the present invention solves the technical
problems underlying the present invention by the provision of a
bioreactor system for the cultivation of mammalian cells
characterised in that a) a first bioreactor with a volume of at
least 500 l is connected with b) a second bioreactor with a volume
of at least 2000 l, which has a volume greater than the first
bioreactor and wherein the second bioreactor with a volume of at
least 2000 l is connected with c) a third bioreactor having at
least two impellers and a volume of at least 10 000 l, which has a
volume greater than the second bioreactor.
[0008] The present invention solves the technical problem
underlying the present invention furthermore by the provision of a
method to cultivate and propagate mammalian cells, characterised in
that a) at least one mammalian cell is cultivated under suitable
conditions and in a suitable culture medium in a first bioreactor
with a volume of at least 500 l, b) the medium containing the cells
obtained by propagation of the at least one mammalian cell is
transferred into a second bioreactor with a volume of at least 2000
l, c) the transferred cells are cultivated in the second bioreactor
with a volume of at least 2000 l, d) the medium containing the
cells obtained in step c) is transferred into a third bioreactor
with a volume of at least 10 000 l and e) the transferred cells are
cultivated in the third bioreactor with a volume of at least 10 000
l.
[0009] According to the invention, the cultivated cells are
eukaryotic cells, preferably animal cells, more preferably
mammalian cells. The mammalian cells can be for example human cell
lines, mouse myeloma (NS0)-cell lines, Chinese hamster ovary
(CHO)-cell lines or hybridoma-cell lines. Preferably the mammalian
cells are CHO-cell lines.
[0010] Preferably the cultivated cells are used to produce
antibodies, more preferably monoclonal antibodies, and/or
recombinant proteins, more preferably recombinant proteins for
therapeutic use. Of course the cells may produce peptides, amino
acids, fatty acids or other useful biochemical intermediates or
metabolites. According to the invention the target concentration of
the proteins produced by the cultivated cells is more than 0.5 g/l,
preferably more than 2.0 g/l and most preferred more than 10.0 g/l.
The method according to the invention can be used as a batch or in
a fed-batch process. Although the cell-culture-medium used in the
method according to the invention is preferably protein free
medium, the design does not exclude the use of protein containing
streams.
[0011] According to the invention a bioreactor is a biocompatible
tank or vessel having additional equipment, for example impellers,
baffles, spargers and/or ports, which specifically allows for the
cultivation and propagation of mammalian cells. Preferably the tank
or vessel is in the form of a tube, having on both ends of the
tube, which build preferably the top and the bottom of the tank,
plates. The plates are called head plate and base plate. In a
particularly preferred embodiment of the present invention the base
plate is an American Society of Mechanical Engineers Flanged and
Dished (ASME F&D) designed base plate. The head-plate design
preferably accommodates a manway or is preferably a flanged head
plate to allow access/removal of the impellers.
[0012] The total tank height is the tangential line from the inner
tank side of the base to the inner tank side of the head of the
tank.
[0013] The freeboard height is defined as the length of straight
side above the liquid head when the bioreactor is filled to it's
operating volume.
[0014] A minimum freeboard height is necessary taking into account
the extent of foam build up during operation, gas hold up at
maximum allowed agitation and aeration and errors in metering
liquid.
[0015] The bioreactor according to the invention has a volume of
preferably at least 500 l, more preferably of at least 1000 l, more
preferably of at least 4000 l, even more preferably of at least 10
000 l, even more preferably of at least 20 000 l. Most preferably
the bioreactor according to the invention has a volume of 1000 l,
1307 l, 4000 l, 5398 l, 20 000 l or 27 934 l.
[0016] Preferably, the bioreactor has a maximum volume of 100 000
l, more preferably the bioreactor has a maximum volume of 50 000 l,
most preferably the bioreactor has a maximum volume of 30 000
l.
[0017] The design of the bioreactors according to the present
invention ensures a homogenous environment with respect to process
parameters such as pH, dissolved oxygen tension (DOT) and
temperature, maintaining a well mixed cell suspension and blending
nutrient feeds within the bioreactor. This provides the necessary
physicochemical environment for optimal cell growth, product
accumulation and product quality. The design of the bioreactors
according to the present invention furthermore ensures the
maintenance of geometric similarity. This allows a scale down model
to be developed at 12 litre laboratory and 500 litre pilot
scales.
[0018] The bioreactor for the cultivation of mammalian cells
according to the invention has at least two impellers. More
preferably, the bioreactor has two impellers, even more preferably
a top impeller and a bottom impeller.
[0019] The bioreactor for the cultivation of mammalian cells
according to the invention has preferably at least one top impeller
and at least one bottom impeller, wherein the top impeller is
preferably a hydrofoil impeller.
[0020] The bioreactor for the cultivation of mammalian cells
according to the invention has preferably at least one top impeller
and at least one bottom impeller, wherein the top impeller is a
hydrofoil impeller.
[0021] The bioreactor for the cultivation of mammalian cells
according to the invention has preferably a volume of at least 1000
l and at least one top impeller and at least one bottom impeller,
wherein the top impeller is a hydrofoil impeller.
[0022] The bioreactor for the cultivation of mammalian cells
according to the invention has preferably a volume of at least 4000
l and at least one top impeller and at least one bottom impeller,
wherein the top impeller is preferably a hydrofoil impeller.
[0023] The bioreactor for the cultivation of mammalian cells
according to the invention has preferably a volume of at least 4000
l and at least one top impeller and at least one bottom impeller,
wherein the top impeller is a hydrofoil impeller.
[0024] In a preferred embodiment of the invention the top impeller
is a hydrofoil impeller. The top impeller can be used preferably to
provide strong bulk mixing.
[0025] In a preferred embodiment of the invention the bottom
impeller is a hydrofoil impeller. In a preferred embodiment of the
invention the top impeller and the bottom impeller are a hydrofoil
impeller.
[0026] In a preferred embodiment of the invention at least the top
impeller is a hydrofoil impeller. In a preferred embodiment of the
invention all impellers are hydrofoil impellers.
[0027] According to a preferred embodiment of the invention the
bottom impeller is a high-solidity pitch-blade impeller or a
high-solidity hydrofoil impeller. The bottom impeller can be used
preferably for the dissipation of sparged gas.
[0028] Preferably, the hydrofoil impellers provide much greater
liquid motion, resulting in a greater bulk-mixing, for a given
amount of power input. This can also depend of the flow number
(N.sub.q).
[0029] Preferably, non-hydrofoil impellers can provide liquid
motion but at greater power inputs. This can have consequences on
the health of shear-sensitive mammalian cells.
[0030] In a preferred embodiment of the invention the hydrofoil
impeller is a down-flowing impeller or a up-flowing impeller.
[0031] In a preferred embodiment of the invention the top impeller
is a down-flowing impeller. In a preferred embodiment of the
invention the top impeller is a down-flowing axial hydrofoil
impeller.
[0032] In a preferred embodiment of the invention the pulling down
characteristics of the top impeller are used to mix the well
aerated liquid surface with the liquid bulk.
[0033] In a preferred embodiment of the invention the hydrofoil
impeller is a high efficiency hydrofoil impeller. In a preferred
embodiment of the invention the hydrofoil impeller is a
Chemineer--model SC-3 impeller, a LIGHTNIN--model A310 or A510
impeller, a Promix--model PHF series impeller or a Cleaveland
Eastern Mixers impeller.
[0034] In a preferred embodiment of the invention the top impeller
is a high efficiency hydrofoil impeller. In a preferred embodiment
of the invention the top impeller is a Chemineer--model SC-3
impeller, a LIGHTNIN--model A310 or A510 impeller, a Promix--model
PHF series impeller or a Cleaveland Eastern Mixers impeller.
[0035] The top impeller is preferably a three-bladed hydrofoil
design impeller, for example a A310-type impeller from LIGHTNIN.
The bottom impeller is preferably a four-pitched-bladed
high-solidity impeller, for example of the A315-type from LIGHTNIN.
The impeller to tank diameter ratio of the top impeller
(D.sub.top/T) and/or of the bottom impeller (D.sub.bottom/T) is
preferably at least 0.35 and at most 0.55, more preferably at least
0.40 and at most 0.48, and most preferably at least 0.44, and at
most 0.46. A diameter greater than 0.5 results in disruption in
axial flow, hence poor agitation and aeration.
[0036] The top impeller power number (N.sub.p) is preferably at
least 0.1 and at most 0.9, more preferably at least 0.25 and at
most 0.35, most preferably 0.3. The top impeller flow number
(N.sub.q) is preferably at least 0.4 and at most 0.9, more
preferably at least 0.50 and at most 0.60, most preferably 0.56.
The bottom impeller power number (N.sub.p) is preferably at least
0.5 and at most 0.9, more preferably at least 0.70 and at most
0.80, most preferably 0.75. The bottom impeller flow number
(N.sub.q) is preferably at least 0.50 and at most 0.85, more
preferably at least 0.70 and at most 0.80, most preferably
0.73.
[0037] The impeller power number (N.sub.p) is a measure of an
impeller efficiency to impart the kinetic energy of the rotating
impeller blades to the fluid. It is important in quantifying the
gas dispersion. The impeller flow number (N.sub.q) is a measure of
pumping ability of the impeller and is important in quantifying
fluid bulk movement.
[0038] The agitation rate of the at least two impellers is
dependent on the scale. However, in a particularly preferred
embodiment of the invention the agitation rate of the at least two
impellers is at most 200 rounds per minute (rpm), more preferably
at most 165 rpm.
[0039] The impeller spacing (D.sub.s) is the space between the at
least two impellers. It is in a particularly preferred embodiment
of the invention at least 1.times. the diameter of the bottom
impeller (D.sub.bottom) and at most 2.times.D.sub.bottom, more
preferably it is 1.229.times.D.sub.bottom or 2.times.D.sub.bottom.
This will allow both impellers to remain submerged at the lowest
post-inoculation volume.
[0040] The liquid height above the upper impeller (D.sub.o) is in a
particularly preferred embodiment of the invention at least
0.3.times. the diameter of the top impeller (D.sub.top) and at most
2.5.times.D.sub.top. More preferably it is at least
0.5.times.D.sub.top and at most 2.0.times.D.sub.top.
[0041] The bottom clearance (D.sub.c) is the clearance between the
tank bottom and the centre-line of the bottom impeller. In a
particularly preferred embodiment of the invention it is at least
0.35.times.D.sub.bottom, more preferably it is either
0.4.times.D.sub.bottom or 0.75.times.D.sub.bottom.
[0042] The design of the impellers in the bioreactor according to
the present invention provides optimal hydrodynamic characteristics
in terms of bulk mixing, gas dispersion and low shear. The
mammalian cells are kept in a homogeneous suspension by agitation
via the impeller system according to the present invention.
[0043] The design of the impellers in the bioreactor according to
the present invention provides rapid mixing, maintain homogeneity,
maintain mammalian cells in suspension and gas bubble dispersion.
The design of the impellers in the bioreactor according to the
present invention minimises cell damage through shear forces
originating from impeller geometry and eddies or vortices created
behind the impeller blades.
[0044] In a particularly preferred embodiment of the present
invention, the at least two impellers are a top driven agitator
system.
[0045] The supply of air, especially compressed air, or specific
gases, preferably oxygen, nitrogen and/or CO.sub.2 is realised
preferably through at least one sparger.
[0046] The bioreactor according to the invention has preferably at
least one sparger, more preferably the bioreactor has one sparger
or two spargers. The bioreactor according to the invention has
preferably two spargers. Preferably the bioreactor has at least one
sparger with a pipe-geometry. Preferably the at least one sparger
is of the flute-type or is a sintered sparger. Preferably the at
least one sparger is of the flute-type. In particularly preferred
embodiment of the present invention a crescent pipe is explored.
The curvature of the crescent is preferably 0.8.times.D.sub.bottom.
In order to aid installation and removal from side ports of the
bioreactor the crescent circumference is preferably 240.degree. of
the complete circumference of 0.8.times.D.sub.bottom ring.
[0047] The at least one sparger provides sufficient oxygen mass
transfer (characterised by K.sub.La) to meet the oxygen demand of
the culture. The at least one sparger provides a K.sub.La up to 20
h.sup.-1 for cultures reaching up to 20.times.10.sup.6 cells per ml
with an oxygen uptake rate of 5 mmol/l per hour. Two spargers used
as a dual sparger system allow the removal of dissolved CO.sub.2
and control of dissolved oxygen tension (DOT). Fluted spargers
offer the benefits of easier cleaning in place (CIP) and
sterilisation in place (SIP), aids with dCO.sub.2 stripping and
reduced operational costs as it is multiple use. Sintered spargers
provide higher K.sub.La values. The lower intrinsic K.sub.La value
with the fluted sparge design can be compensated by the use of
oxygen enriched air. The gas flow rates are scaled up on the basis
of constant superficial gas velocity.
[0048] It is important in large scale cultivation of mammalian
cells to maintain a homogenous physicochemical environment in terms
of dissolved oxygen, culture pH, and temperature, and dissolved
CO.sub.2, nutrient and metabolite concentration gradients. Whilst
ensuring the physicochemical environment is homogenous through
using appropriate agitation and aeration, it is important to ensure
the selected operating agitation and aeration conditions do not
produce adverse shear environment. The appropriate balance between
ensuring homogenous environment that will promote good cell growth
and productivity of mammalian cell culture processes whilst
minimising the adverse effects of shear environment is dealt with
in this invention. This is achieved through prescribing specific
bioreactor geometries, impeller design and positioning, sparger
design and positioning and specific operating limits for agitation
and aeration rates.
[0049] The major damage to mammalian cells in stirred and sparged
bioreactors comes from interfacial shear. Interfacial shear occurs
as sparged gas bubbles coalesce and burst [ref: Ma N, Koelling K W,
Chalmers J J. Biotechnol Bioeng. 2002 Nov. 20; 80(4):428-37.
Erratum in: Biotechnol Bioeng. 2003 Feb. 5; 81(3):379]. Thus
minimising sparged gas flows and excessive build up of foam is
desirable. The interfacial shear can be minimised through a
combination of approaches first by promoting surface aeration
through good mixing of the liquid surface with the liquid bulk and
secondly higher oxygen driving force by segregated oxygenation of
cultures through the preferably two spargers.
[0050] The prescribed positioning of the hydrofoil impeller,
particularly the liquid height above the upper impeller, D.sub.O
preferably being around 0.5.times.D.sub.top, below the liquid
surface can aid strong and continuous exchange of the liquid
surface with the liquid bulk thereby mixing the well oxygenated
liquid surface with less oxygenated liquid bulk. The prescribed
impeller spacing, preferably being D.sub.s=1.times.D.sub.bottom to
2.times.D.sub.bottom can permit the down-flow of liquid generated
by the upper impeller to feed fluid flow into the lower impeller
thereby ensuring the whole fluid bulk is well-mixed and separate
mixing zones are not made. The prescribed impeller bottom
clearance, preferably being D.sub.c=0.35.times.D to 0.75.times.D
can ensure that the bulk flow is able to deflect off the curved
ASME F&D base and rise upwards along the walls of the
bioreactor.
[0051] The segregation of the `on-demand` oxygenated sparged gas
through the control sparger from the non-oxygenated sparged gases
(such as CO.sub.2, air and nitrogen ballasts) through a ballast
sparger can allow greater residence time and path length of highly
oxygenated sparge gas bubbles in the fluid bulk before disengaging
out of the fluid bulk and into the headspace. This can permit
greater oxygen transfer rates to be provided for a given volumetric
mass transfer coefficient, k.sub.La. The residence time and path
length of the sparged gas bubbles can be extended further through
specifying down-flowing axial hydrofoil impellers that continuingly
pull the liquid surface and liquid bulk down.
[0052] The bioreactor according to the invention has preferably at
least one baffle, more preferably at least two baffles. The
bioreactor according to the invention has most preferably four
baffles.
[0053] Baffles are vertical radially located plates. Baffles are
used to prevent the formation of a funnel or vortex formation.
[0054] In a preferred embodiment of the invention, the length of
the at least one baffle is 1.1.times. the total straight height (H)
of the bioreactor. The width of the baffle (W) is preferably
0.1.times. the internal diameter of the tank (T). The baffle
clearance (W.sub.c) is preferably 0.01.times. the internal diameter
of the tank (T). The height of at least one baffle (H.sub.baffle)
is preferably 1.1.times. the total straight height (H)--the height
of the bioreactor-head (H.sub.h). Therefore H.sub.baffle is
preferably calculated according to the formula
H.sub.baffle=1.1.times.H-H.sub.h.
[0055] The thickness of the at least one baffle is not specified
but the thickness needs to ensure rigidity to the radial component
of the fluid flow. Additionally thickness needs to ensure the
baffle plates are not warped during SIP thereby affecting the
baffle to tank wall clearance.
[0056] The bioreactor according to the invention has preferably at
least two ports for alkali addition. More preferably, the
bioreactor has two ports for alkali addition. Most preferably, the
bioreactor has two ports for alkali addition, wherein the first
port is located at the central line of the bottom impeller and the
second port is located at the central line of the top impeller.
Preferably the pH probes are located diametrically opposite the
alkali addition points into the bioreactor.
[0057] In a preferred embodiment of the invention, the bioreactor
has a volume of 1000 l. The head volume (V.sub.h) of a 1000 l
bioreactor is preferably at least 45 l and at most 65 l, more
preferably the head volume is 55 l. The base volume (V.sub.b) of
the 1000 l bioreactor is preferably at least 45 l and at most 65 l,
more preferably the base volume is 55 l. The tank internal diameter
(T) of the 1000 l bioreactor according to the invention is
preferably at least 850 mm and at most 900 mm, more preferably the
tank internal diameter is 864 mm. The tank cross-sectional area (A)
of the 1000 l bioreactor according to the invention is preferably
at least 0.55 m.sup.2 and at most 0.65 m.sup.2, more preferably the
tank cross-sectional area is 0.586 m.sup.2. The head height
(H.sub.h), which is the height of the head-plate, and/or the base
height (H.sub.b), which is the height of the base-plate, of the
bioreactor with a volume of 1000 l according to the invention is
preferably at least 120 mm and at most 180 mm, more preferably the
head height and/or the base height is 151 mm. The total tank height
of the 1000 l bioreactor according to the invention is preferably
at least 2000 mm and at most 2600 mm, more preferably the total
tank height is 2347 mm. The top impeller diameter (D.sub.top)
and/or the bottom impeller diameter (D.sub.bottom) of the 1000 l
bioreactor according to the invention is preferably at least 350 mm
and at most 400 mm, more preferably the top impeller diameter
and/or the bottom impeller diameter is 381 mm. The clearance
between the tank bottom and centre-line of the bottom impeller
(D.sub.c) is for the 1000 l bioreactor according to the invention,
preferably at least 120 mm and at most 180 mm, more preferably the
clearance is 152 mm. The distance between the at least two
impellers, also known as impeller separation (D.sub.s) is for the
1000 l bioreactor, according to the invention, preferably at least
730 and at most 790 mm, more preferably the impeller separation is
762 mm.
[0058] The impeller shaft diameter for the 1000 l bioreactor
according to the invention is preferably at least 102 mm and at
most 152 mm. If the 1000 l bioreactor according to the invention
has baffles, the length of the baffles is preferably at least 2000
mm and at most 2400 mm, more preferably the length is 2250 mm. The
width of the baffles for the 1000 l bioreactor according to the
invention is preferably at least 70 mm and at most 100 mm, more
preferably the width is 86 mm. The baffle clearance for the 1000 l
bioreactor according to the invention is preferably at least 7 mm
and at most 11 mm, more preferably the baffle clearance is 9 mm.
The baffle height (H.sub.baffle) for the 1000 l bioreactor
according to the invention is preferably at least 2000 mm and at
most 2200 mm, more preferably the baffle height is 2099 mm. The
1000 l bioreactor according to the invention has preferably at
least one sparger, more preferably it has one sparger. The at least
one sparger of the 1000 l bioreactor according to the invention has
preferably an orifice- or pore-size of at least 1.5 mm and at most
2.5 mm, more preferably the orifice- or pore-size is 2 mm. The
orifice- or pore-number is preferably at least 20 and at most 40,
more preferably the orifice- or pore-number is 30. The sparger
length (S.sub.L is preferably at least 150 mm and at most 550 mm,
more preferably the sparger length is 305 mm. The sparger to tank
bottom clearance (S.sub.c) of the 1000 l bioreactor according to
the invention is preferably at least 50 mm and at most 75 mm, more
preferably the sparger to tank bottom clearance is 64 mm. The
sparger to bottom impeller clearance (D.sub.c-S.sub.c) of the 1000
l bioreactor according to the invention is preferably at least 75
mm and at most 100 mm, more preferably the sparger to bottom
impeller clearance is 88 mm.
[0059] In a preferred embodiment of the invention, the bioreactor
has a volume of 4000 l. The head volume (V.sub.h) of a 4000 l
bioreactor is preferably at least 340 l and at most 370 l, more
preferably the head volume is 359 l. The base volume (V.sub.b) of
the 4000 l bioreactor is preferably at least 340 l and at most 370
l, more preferably the base volume is 359 l. The tank internal
diameter (T) of the 4000 l bioreactor according to the invention is
preferably at least 1600 mm and at most 1650 mm, more preferably
the tank internal diameter is 1626 mm. The tank cross-sectional
area (A) of the 4000 l bioreactor according to the invention is
preferably at least 1.90 m.sup.2 and at most 2.30 m.sup.2, more
preferably the tank cross-sectional area is 2.076 m.sup.2. The head
height (H.sub.h) and/or the base height (H.sub.b) of the bioreactor
with a volume of 4000 l according to the invention is preferably at
least 260 mm and at most 300 mm, more preferably the head height
and/or the base height is 282 mm. The total tank height of the 4000
l bioreactor according to the invention is preferably at least 2300
mm and at most 3100 mm, more preferably the total tank height is
2817 mm. The top impeller diameter (D.sub.top) and/or the bottom
impeller diameter (D.sub.bottom) of the 4000 l bioreactor according
to the invention is preferably at least 680 mm and at most 740 mm,
more preferably the top impeller diameter and/or the bottom
impeller diameter is 710 mm. The clearance between the tank bottom
and centre line of the bottom impeller (D.sub.c) is for the 4000 l
bioreactor according to the invention, preferably at least 500 mm
and at most 560 mm, more preferably the clearance is 531 mm. The
distance between the at least two impellers, also known as impeller
separation (D.sub.s) is for the 4000 l bioreactor, according to the
invention, preferably at least 840 mm and at most 900 mm, more
preferably the impeller separation is 872 mm. The impeller shaft
diameter for the 4000 l bioreactor according to the invention is
preferably at least 51 mm and at most 64 mm. If the 4000 l
bioreactor according to the invention has baffles, the length of
the baffles is preferably at least 2200 mm and at most 2600 mm,
more preferably the length is 2477 mm. The width of the baffles for
the 4000 l bioreactor according to the invention is preferably at
least 150 mm and at most 180 mm, more preferably the width is 163
mm.
[0060] The baffle clearance is for the 4000 l bioreactor according
to the invention preferably at least 12 mm and at most 20 mm, more
preferably the baffle clearance is 16 mm. The baffle height
(H.sub.baffle) for the 4000 l bioreactor according to the invention
is preferably at least 2100 mm and at most 2300 mm, more preferably
the baffle height is 2195 mm. The 4000 l bioreactor according to
the invention has preferably at least one sparger, more preferably
it has one sparger. The at least one sparger of the 4000 l
bioreactor according to the invention has preferably an orifice- or
pore-size of at least 1.5 mm and at most 2.5 mm, more preferably
the orifice- or pore-size is 2 mm. The orifice- or pore-number for
the 4000 l bioreactor according to the invention is preferably at
least 80 and at most 120, more preferably the orifice- or
pore-number is 100. The sparger length (S.sub.L) is preferably at
least 250 mm and at most 800 mm, more preferably the sparger length
is 568 mm. The sparger to tank bottom clearance (S.sub.c) of the
4000 l bioreactor according to the invention is preferably at least
315 mm and at most 360 mm, more preferably the sparger to tank
bottom clearance is 337 mm. The sparger to bottom impeller
clearance (D.sub.c-S.sub.c) of the 1000 l bioreactor according to
the invention is preferably at least 180 mm and at most 205 mm,
more preferably the sparger to bottom impeller clearance is 194
mm.
[0061] In a preferred embodiment of the invention, the bioreactor
has a volume of 20 000 l. The head volume (V.sub.h) of a 20 000 l
bioreactor is preferably at least 1600 l and at most 2000 l, more
preferably the head volume is 1803 l. The base volume (V.sub.b) of
the 20 000 l bioreactor is preferably at least 1600 l and at most
2000 l, more preferably the base volume is 1803 l. The tank
internal diameter (T) of the 20 000 l bioreactor according to the
invention is preferably at least 2500 mm and at most 3000 mm, more
preferably the tank internal diameter is 2794 mm. The tank
cross-sectional area (A) of the 20 000 l bioreactor according to
the invention is preferably at least 5.8 m.sup.2 and at most 6.5
m.sup.2, more preferably the tank cross-sectional area is 6.131
m.sup.2. The head height (H.sub.h) and/or the base height (H.sub.b)
of the bioreactor with a volume of 20 000 l according to the
invention is preferably at least 460 mm and at most 500 mm, more
preferably the head height and/or the base height is 485 mm. The
total tank height of the 20 000 l bioreactor according to the
invention is preferably at least 4800 mm and at most 5100 mm, more
preferably the total tank height is 4933 mm. The top impeller
diameter (D.sub.top) and/or the bottom impeller diameter
(D.sub.bottom) of the 20 000 l bioreactor according to the
invention is preferably at least 1100 mm and at most 1300 mm, more
preferably the top impeller diameter and/or the bottom impeller
diameter is 1219 mm. The clearance between the tank bottom and
centre line of the bottom impeller (D.sub.c) is for the 20 000 l
bioreactor according to the invention, preferably at least 890 mm
and at most 945 mm, more preferably the clearance is 913 mm. The
distance between the at least two impellers, also known as impeller
separation (D.sub.s) is for the 20 000 l bioreactor, according to
the invention, preferably at least 1200 mm and at most 1700 mm,
more preferably the impeller separation is 1498 mm. The impeller
shaft diameter for the 20 000 l bioreactor according to the
invention is preferably at least 51 mm and at most 64 mm. If the 20
000 l bioreactor according to the invention has baffles, the length
of the baffles is preferably at least 4000 mm and at most 4600 mm,
more preferably the length is 4365 mm. The width of the baffles for
the 20 000 l bioreactor, according to the invention, is preferably
at least 260 mm and at most 290 mm, more preferably the width is
279 mm. The baffle clearance for the 20 000 l bioreactor, according
to the invention, is preferably at least 20 mm and at most 35 mm,
more preferably the baffle clearance is 28 mm. The baffle height
(H.sub.baffle) for the 20 000 l bioreactor according to the
invention is preferably at least 3600 mm and at most 4050 mm, more
preferably the baffle height is 3882 mm. The 20 000 l bioreactor
according to the invention has preferably at least one sparger,
more preferably it has two spargers. If the 20 000 l bioreactor
according to the invention has two spargers one is preferably a
control sparger and one is preferably a ballast sparger. The
control sparger for the 20 000 l bioreactor according to the
invention has preferably an orifice- or pore-size of at least 3 mm
and at most 5 mm, more preferably the orifice- or pore-size is 4
mm. The ballast sparger for the 20 000 l bioreactor according to
the invention has preferably an orifice- or pore-size of at least 5
mm and at most 7 mm, more preferably the orifice- or pore-size is 6
mm. The orifice/pore number of the control sparger for the 20 000 l
bioreactor according to the invention is preferably at least 230
and at most 270, more preferably the orifice- or pore-number is
250. The orifice-pore-number of the ballast sparger for the 20 000
l bioreactor according to the invention is preferably at least 85
and at most 115, more preferably the orifice- or pore-number is
100. The sparger length (S.sub.L) for the control and/or the
ballast sparger is preferably at least 500 mm and at most 2000 mm,
more preferably the sparger length is 1077 mm. The sparger to tank
bottom clearance (S.sub.c) of the 20 000 l bioreactor according to
the invention is preferably for the control and/or the ballast
sparger at least 560 mm and at most 620 mm, more preferably the
sparger to tank bottom clearance is 593 mm. The sparger to bottom
impeller clearance (D.sub.c-S.sub.c) of the 20 000 l bioreactor
according to the invention is for the control and/or the ballast
sparger preferably at least 300 mm and at most 340 mm, more
preferably the sparger to bottom impeller clearance is 320 mm. The
requirement to add ballast from a separate sparger, the ballast
sparger, prevents dilution of oxygen or oxygen enriched DOT demand
gas with the ballast gas. This ensures the best oxygen transfer
rate (OTR), as the oxygen concentration gradient of the bubbles
emerging from the sparger is greatest. Secondly, the use of a
ballast sparger allows spargers to be located at different
positions to avoid impacting DOT control on delivering desired
ballast for pCO.sub.2 control. The ballast sparger can be
independently designed from the control sparger.
[0062] With the bioreactor design according to the invention,
different subculture ratios can be performed. In a particularly
preferred embodiment the subculture ratios performed are subculture
ratios of at least 1 in 5 (20% v/v) and at most 1 in 9 (11% v/v),
more preferred 1 in 5 (20% v/v) or 1 in 9 (11% v/v).
[0063] The invention also includes a method to cultivate and
propagate mammalian cells, characterised in that at least one
mammalian cell is cultivated under suitable conditions and in a
suitable culture medium in a bioreactor according the
invention.
[0064] Bioreactors according to the invention include all
bioreactors having at least two impellers and showing at least one
feature or a combination of different features outlined above.
[0065] In the method according to the invention, the agitation rate
of the at least two impellers of the bioreactor is preferably at
least 55 W/m.sup.3 and at most 85 W/m.sup.3. Preferably, air is
sparged into the culture medium with a speed of at least
5.times.10.sup.-5 m/s, more preferably of at least
10.times.10.sup.-5 m/s.
[0066] In a particularly preferred embodiment of the present
invention alkali is added through two addition ports to distribute
the alkali, which are, preferably spatially separated from each
other. This ensures quicker blending of alkali in the event of long
re-circulation time in the tank. CO.sub.2 is preferably added via a
control sparger.
[0067] Alkali and/or CO.sub.2 are preferably used to regulate the
pH of the culture-medium.
[0068] It is preferred that control and back-up probes be in the
lower port ring at 913 mm from tank bottom.
[0069] In a preferred embodiment of the present invention, the
method according to the invention takes place in a bioreactor with
a volume of 1000 l. The volume of the culture medium used in the
method using a 1000 l bioreactor is preferably during the
pre-inoculation 50 l to 250 l. During the post-inoculation the
volume of the culture medium is preferably at least 300 l and at
most 960 l. In the pretransfer/harvest phase, the volume of the
culture medium in the 1000 l bioreactor is preferably at least 300
l and at most 960 l. The minimum operating volume (V.sub.min) in a
bioreactor with the volume of 1000 l according to the invention is
preferably between 80 l and 120 l, more preferably the minimum
operating volume is 100 l, the maximum operating volume (V) is
preferably at least 900 l and at most 1100 l, the maximum operating
volume is more preferably 1000 l. The minimum stirred volume is
preferably at least 230 l and at most 255 l, more preferably the
minimum stirred volume is 245 l. The liquid height at the minimum
operating volume (H.sub.min) is in a bioreactor with a volume of
1000 l preferably at least 210 mm and at most 240 mm, more
preferably the liquid height at the minimum operating volume is 228
mm. The liquid height at the maximum operating volume (H.sub.L) in
a bioreactor with a volume of 1000 l is preferably at least 1500 mm
and at most 1900 mm, more preferably the liquid height at the
maximum operating volume is 1764 mm. The minimum aspect ratio
(H.sub.min/T) is preferably at least 0.15 and at most 0.19, more
preferably the minimum aspect ration is 0.17. The maximum aspect
ratio (H.sub.L-T) for the bioreactor with a volume of 1000 l used
in a method according to the invention is preferably at least 1.8
and at most 2.1, more preferably the maximum aspect ratio is 1.96.
The freeboard volume is preferably at least 270 l and at most 310
l, more preferably the freeboard volume is 293 l. The freeboard
height is preferably at least 450 mm and at most 550 mm, more
preferably the freeboard height is 500 mm. The total straight
height (H) is preferably at least 1900 mm and at most 2200 mm, more
preferably the total straight height is 2045 mm. The height of the
upper probe- or sample-ring is preferably at least 900 mm and at
most 1200 mm, more preferably the height of the upper probe- or
sample-ring is 1093 mm. The height of the lower probe-sample ring
is preferably at least 152 mm and at most 286 mm.
[0070] In a preferred embodiment of the present invention, the
method according to the invention takes place in a bioreactor with
a volume of 4000 l. The volume of the culture medium used in the
method using a 4000 l bioreactor is preferably during the
pre-inoculation 1914 l to 3077 l. During the post-inoculation the
volume of the culture medium is preferably at least 2153 l and at
most 3846 l. In the pretransfer/harvest phase, the volume of the
culture medium in the 4000 l bioreactor is preferably at least 2153
l and at most 3846 l. The minimum operating volume (V.sub.min) in a
bioreactor with the volume of 4000 l according to the invention is
preferably between 1500 l and 2200 l, more preferably the minimum
operating volume is 1900 l, the maximum operating volume (V) is
preferably at least 3800 l and at most 4200 l, the maximum
operating volume is more preferably 4000 l. The minimum stirred
volume is preferably at least 1500 l and at most 1800 l, more
preferably the minimum stirred volume is 1654 l. The liquid height
at the minimum operating volume (H.sub.min) is in a bioreactor with
a volume of 4000 l preferably at least 800 mm and at most 1200 mm,
more preferably the liquid height at the minimum operating volume
is 1024 mm. The liquid height at the maximum operating volume
(H.sub.L) in a bioreactor with a volume of 4000 l is preferably at
least 1800 mm and at most 2200 mm, more preferably the liquid
height at the maximum operating volume is 2034 mm. The minimum
aspect ratio (H.sub.min/T) is preferably at least 0.55 and at most
0.75, more preferably the minimum aspect ration is 0.63. The
maximum aspect ratio (H.sub.L-T) for the bioreactor with a volume
of 4000 l used in a method according to the invention is preferably
at least 1.1 and at most 1.4, more preferably the maximum aspect
ratio is 1.25. The freeboard volume is preferably at least 850 l
and at most 1250 l, more preferably the freeboard volume is 1039 l.
The freeboard height is preferably at least 450 mm and at most 550
mm, more preferably the freeboard height is 500 mm. The total
straight height (H) is preferably at least 2000 mm and at most 2400
mm, more preferably the total straight height is 2252 mm. The
height of the upper probe- or sample-ring is preferably at least
1200 mm and at most 1600 mm, more preferably the height of the
upper probe- or sample-ring is 1403 mm. The height of the lower
probe- or sample-ring is preferably at least 500 mm and at most 550
mm, more preferably the height of the lower probe- or sample-ring
is 531 mm.
[0071] In a preferred embodiment of the present invention, the
method according to the invention takes place in a bioreactor with
a volume of 20 0001. The volume of the culture medium used in the
method using a 20 000 l bioreactor is preferably during the
pre-inoculation 13 913 l to 17 096 l. During the post-inoculation
the volume of the culture medium is preferably at least 17 391 l
and at most 19 231 l. In the pretransfer/harvest phase, the volume
of the culture medium in the 20 000 l bioreactor is preferably at
least 20 000 l and at most 21 739 l. The minimum operating volume
(V.sub.min) in a bioreactor with the volume of 20 000 l according
to the invention is preferably between 9000 l and 16 0001, more
preferably the minimum operating volume is 13 0001, the maximum
operating volume (V) is preferably at least 19 0001 and at most 25
000 l, the maximum operating volume is more preferably 22 000 l.
The minimum stirred volume is preferably at least 8100 l and at
most 8500 l, more preferably the minimum stirred volume is 8379 l.
The liquid height at the minimum operating volume (H.sub.min) is in
a bioreactor with a volume of 20 000 l preferably at least 2100 mm
and at most 2500 mm, more preferably the liquid height at the
minimum operating volume is 2309 mm. The liquid height at the
maximum operating volume (H.sub.L) in a bioreactor with a volume of
20 000 l is preferably at least 3550 mm and at most 3950 mm, more
preferably the liquid height at the maximum operating volume is
3777 mm. The minimum aspect ratio (H.sub.min/T) is preferably at
least 0.70 and at most 0.99, more preferably the minimum aspect
ration is 0.83. The maximum aspect ratio (H.sub.L-T) for the
bioreactor with a volume of 20 000 l used in a method according to
the invention is preferably at least 1.2 and at most 1.5, more
preferably the maximum aspect ratio is 1.35. The freeboard volume
is preferably at least 5750 l and at most 6500 l, more preferably
the freeboard volume is 6131 l. The freeboard height is preferably
at least 900 mm and at most 1100 mm, more preferably the freeboard
height is 1000 mm. The total straight height (H) is preferably at
least 3700 mm and at most 4100 mm, more preferably the total
straight height is 3968 mm. The height of the upper probe- or
sample-ring is preferably at least 2200 mm and at most 2650 mm,
more preferably the height of the upper probe- or sample-ring is
2411 mm. The height of the lower probe- or sample-ring is
preferably at least 880 mm and at most 940 mm, more preferably the
height of the lower probe- or sample-ring is 913 mm.
[0072] For a bioreactor with a volume of 20 000 l the preferred
seeding ratio used is 11% v/v (1 in 9 dilution) or 20% v/v (1 in 5
dilution), with a preferred feed application of 4% v/v to 25% v/v
of the post-inoculation volume. The post-inoculation volume in the
20 000 l bioreactor is preferably adjusted for feed applications up
to 15% such that after the addition of all the feeds the final
volume at harvest ends up at 20 000 l. However, for feed
applications greater then 15% v/v the post-inoculation volume is
preferably adjusted for a 15% v/v feed but following the
application of feeds the final pre-harvest volume will be a minimum
of 20 000 l and a maximum 22 000 l. The 20 000 l bioreactor is
expected to hold a total of 20 000 l to 22 000 l at the end of a
batch.
[0073] The bioreactor with a volume of 20 000 l is preferably
operated in batch or fed batch mode for 10 to 15 days.
[0074] The invention also includes a bioreactor system for the
cultivation of mammalian cells characterised in that a) a first
bioreactor with a volume of at least 500 l, preferably of at least
1000 l, is connected with b) a second bioreactor with a volume of
at least 2000 l, preferably of at least 4000 l, which has a volume
greater than the first bioreactor and wherein the second bioreactor
with a volume of at least 2000 l, preferably of at least 4000 l, is
connected with c) a third bioreactor according to the invention
having a volume of at least 10 000 l, preferably of at least 20 000
l, which has a volume greater than the second bioreactor.
[0075] In a preferred embodiment of the invention, the bioreactor
system is characterised in that at least one of the bioreactors is
a bioreactor according to the invention. More preferably, all of
the bioreactors of the bioreactor system are bioreactors according
to the invention.
[0076] Bioreactors according to the invention are in this context
all bioreactors described in this description, in the examples and
in the claims.
[0077] The bioreactor system according to the invention is also
called bioreactor train or device.
[0078] The bioreactor train comprises preferably different
bioreactors, which are also called stage. The bioreactor with a
volume of at least 500 l, preferably of at least 1000 l corresponds
to stage N-3 and/or N-2. The bioreactor with a volume of at least
2000 l, preferably of at least 4000 l corresponds to stage N-1. The
bioreactor with a volume of at least 10 000 l, preferably of at
least 20 000 l corresponds to stage N.
[0079] The design of the bioreactor train is based on the need to
ensure a homogenous environment with respect to process parameters
such as pH, dissolved oxygen tension (DOT) and temperature,
maintaining a well mixed cell suspension and blending nutrient
feeds within the bioreactor. The bioreactors of the bioreactor
train preferably show geometric similarity. This allows a
scale-down model to develop, for example at 12 l laboratory scales
or 500 l pilot scales. The bioreactors of the stages N-3, N-2 and
N-1 are used as seed-bioreactors. Bioreactor of stage N is used as
a production-bioreactor. The design of the seed- and
production-bioreactors is preferably based on the same principles.
However, some departures can be required to allow for flexibility
in processing.
[0080] In a preferred embodiment of the invention, the aspect ratio
H.sub.L/T is at least 0.17 and at most 1.96.
[0081] In a preferred embodiment of the invention there is a
further bioreactor, especially a 50 l bioreactor corresponding to
stage N-4.
[0082] In a preferred embodiment of the invention, the N-4
bioreactor is a S-200 seed wave bioreactor or a 100 l stirred tank
reactor
[0083] In a preferred embodiment of the invention, liquids, for
example culture medium, can be transported from one bioreactor to
another bioreactor by pneumatic assisted flow or by peristaltic
pumps.
[0084] The invention also includes a method to cultivate and
propagate mammalian cells, characterised in that a) at least one
mammalian cell is cultivated under suitable conditions and in a
suitable culture medium in a first bioreactor with a volume of at
least 500 l, preferably with a volume of at least 1000 l, b) the
medium containing the cells obtained by propagation from the at
least one mammalian cell is transferred into a second bioreactor
with a volume of at least 2000 l, preferably with a volume of at
least 4000 l, c) the transferred cells are cultivated in the second
bioreactor with a volume of at least 2000 l, preferably with a
volume of at least 4000 l, d) the medium containing the cells
obtained in step c) is transferred into a third bioreactor with a
volume of at least 10 000 l, preferably with a volume of at least
20 000 l, and e) the transferred cells are cultivated in the third
bioreactor with a volume of at least 10 000 l, preferably with a
volume of at least 20 000 l.
[0085] In a preferred embodiment of the invention, the method is
characterised in that at least one of the bioreactors used is a
bioreactor according to the invention, more preferably all
bioreactors used are bioreactors according to the invention.
[0086] Bioreactors according to the invention are in this context
all bioreactors described in this description, in the examples and
in the claims.
[0087] The bioreactor of step e) is preferably operated in batch or
fed batch mode. The cells are cultivated in step e) preferably for
10 to 15 days.
[0088] Step a) is also called stage N-3 and/or N-2. Step c) is also
called stage N-1. Step e) is also called stage N.
[0089] Preferably the cultivation conditions in the bioreactors of
steps a), c) and e) are the same. More preferably, the cultivation
conditions in the bioreactors of steps a), c) and e) have a
homogenous environment with respect to process parameters such as
pH, dissolved oxygen tension and temperature. Preferably pH,
dissolved oxygen tension and temperature in the bioreactors of
steps a), c) and e) are the same.
[0090] In a preferred embodiment of the invention, the seeding
ratio after the transfer steps b) and/or d) is at least 10% v/v,
more preferably at least 11% v/v (1 in 9 dilution) and at most 30%
v/v, more preferably 20% v/v (1 in 5 dilution).
[0091] Preferably either the total medium or only a part of the
medium are transferred in steps b) and d).
[0092] Further preferred embodiments of the present invention are
the subject-matter of the sub claims.
[0093] The present invention is illustrated in more detail in the
following examples and the accompanying figures.
[0094] FIG. 1 shows a bioreactor according to the invention. 1 is
the bioreactor. 10 is the diameter of the tank (T). 20 is the total
straight height of the bioreactor (H). 30 is the base height of the
bioreactor (H.sub.b). 40 is the head height of the bioreactor
(H.sub.h). 50 is the liquid height at the maximum operating volume
(H.sub.L). 60 is the top impeller diameter (D.sub.top). 68 is the
top impeller. 70 is the bottom impeller diameter
(D.sub.bottom).sub.. 78 is the bottom impeller. 80 is the clearance
between tank bottom and centre line of the bottom impeller
(D.sub.c). 90 is the impeller separation (D.sub.s). 100 is the
clearance of the top impeller below the liquid surface (D.sub.o).
108 is a sparger. 110 is the sparger to tank bottom clearance
(S.sub.c). 120 is the sparger to bottom impeller clearance
(D.sub.c-S.sub.c). 128 is a baffle. 138 is a port located at the
lower ring. 148 is a port located at the centre-line of the top
impeller 68.
[0095] FIG. 2 shows a bioreactor system of the present invention.
111 is a bioreactor with a volume of 1000 l. 11 is a bioreactor
with a volume of 4000 l. 1 is a bioreactor according to the
invention with a volume of 20 000 l.
EXAMPLE 1
20 000 l Bioreactor
[0096] The 20 000 l bioreactor is operated in batch and fed batch
mode for 10 to 15 days for the cultivation of mammalian cells. The
mammalian cells are kept in a homogeneous suspension by agitation
via an impeller system.
Vessel Geometry
[0097] The vessel geometry for the 20 000 litre bioreactor was
determined by an iterative design basis in which the maximum
working volume, freeboard straight side distance, aspect ratio
H.sub.L/T and impeller to tank diameter, D/T ratio are altered
until an acceptable aspect ratio is achieved.
Bioreactor Aspect Ratio
[0098] This critical design parameter allows characterisation of
bioreactor geometry. Tanks with higher aspect ratio offer longer
gas residence time allowing greater K.sub.La. However increased
head pressure can cause build up of soluble gases. Smaller aspect
ratio H.sub.L/T in tanks can lead to shorter gas residence time
requiring greater gas flow for aeration resulting in greater foam
build up. Impeller driven agitation to increase K.sub.La is also
limited by H.sub.L/T as surface breakage and vortex creation will
occur at lower impeller revolutions in a low aspect ratio. Thus
choice of aspect ratio is largely experience based with some
thought on issues highlighted in table 1.
TABLE-US-00001 TABLE 1 Summary of effect of varying aspect ratio
Process factor High aspect ratio Low aspect ratio Radial mixing
More effective Less effective Mixing time Higher Lower Oxygen
transfer rate Determined by dissolved Determined by dissolved
oxygen control oxygen control Gas flow rate Lower Higher Cell
damage Less More Carbon dioxide Less effective More effective
stripping Pressure variations Higher Lower Ease of scale More
difficult away from More difficult away from up/scale down
currently used aspect currently used aspect (access to scale ratios
ratios data) Cleanability Not affected directly by Not affected
directly by aspect ratio aspect ratio Volume flexibility Less
More
[0099] Table 2 describes the aspect ratios in the 20 000 litre
bioreactor at various operating volumes during normal processing.
The aspect ratios have been tested at 500 litre scale and provided
the superficial gas velocity and power per unit volume are kept
constant the K.sub.La remains constant.
TABLE-US-00002 TABLE 2 Key operating volumes and aspect ratios in
the 20000 litre bioreactor Volume, L Liquid head, mm Aspect ratio,
H.sub.L/T Pre-Inoculation 13913-17096 2458-2977 0.88-1.07 Post
Inoculation 17391-19231 3025-3325 1.08-1.19 Harvest 20000-21739
3451-3734 1.23-1.34
Tank Diameter
[0100] The tank diameter is altered to obtain the optimal aspect
ratio H.sub.L/T. Changes to tank internal diameter (ID) are limited
by acceptable aspect ratio and plant footprint. The ID is 2.794
m.
Tank Height
[0101] Tank height is determined from the maximum operating volume,
aspect ratio H.sub.L/T, freeboard straight side length, base and
top plate design. The final tank height is a compromise value
determined from volumetric contingency for foam, plant height and
impeller shaft length. The tank height from base to head tan line
is 4.933 m.
Freeboard Height
[0102] The freeboard height is defined as the length of straight
side above the liquid head when the bioreactor is filled to it's
maximum operating volume. This is determined by taking into account
the extent of: [0103] Foam build up during operation. [0104] Gas
hold up at maximum allowed agitation and aeration. [0105] Errors in
metering liquid.
[0106] In absence of knowing the exact contribution of each with
piloting the process at full scale an estimate is usually made. The
amount of freeboard height is balanced with the desire to reduce
the impeller shaft length for a top-driven system, where extra
length can complicate the design and selection of available
mechanical seals, the requirement for steady bearing or stabilising
impeller rings. A minimum freeboard height of 1000 mm (or 6100
litre volumetric capacity or 28% v/v of the maximum operating
volume) is therefore used.
Head and Base Plate
[0107] The selection of head and base plate design was made with a
consideration for desired mechanical strength, free draining clean
design and fluid flow. Maintaining consistent plate design between
scale down and full scale will contribute towards maintaining
geometric similarity. The base plate is of American Society of
Mechanical Engineers Flanged and Dished (ASME F&D) design. The
head-plate design accommodates a manway or a flanged head plate to
allow access/removal of the impellers.
Bioreactor Agitation Requirement
[0108] The agitation of the bioreactor is to achieve rapid mixing,
maintain homogeneity, maintain mammalian cells in suspension and
gas bubble dispersion. The underlying issue with achieving the
above objectives is minimising cell damage through shear forces
originating from impeller geometry and eddies or vortices created
behind the impeller blades. A compromise of the above objectives
can be achieved by selection of an appropriate impeller type.
Bottom Versus Top Driven Impeller Shaft
[0109] The decision to drive the agitator shaft from the top or the
bottom of the bioreactor is important and is determined following a
review of a number of issues highlighted in table 3.
TABLE-US-00003 TABLE 3 Key design issues for selection of top
versus bottom entry of impeller shaft Top entry Bottom entry Shaft
Length Long Short Shaft Weight High Low Shaft Diameter Larger
Smaller Impeller shaft on-site Greater plant Less plant height
installation and removal height for servicing and repair Exposure
of cell culture to No exposure Exposure moving and stationary seal
faces .sup.1Pressurization between Lower Higher due to the liquid
seal and vessel head Seal Lubricant leakage rate Lower Higher Base
plate Design Simple Complex Sparger to tank bottom Unrestricted
Restricted positioning CIP validation Simple Complicated by sub-
merged mechanical seal Scale up and scale down Consistent with
Inconsistent with lab and consistency lab and pilot pilot scale
scale .sup.1Pressure differential between seal and bioreactor
critical for lubrication and cooling.
[0110] Top-entry impeller shafts tend to be longer than
bottom-entry, which results in the shaft being heavier and larger
diameter. Additionally the shaft length together with the inherent
clearance between the two faces of the mechanical seal may dictate
the requirement for steady bearings or stabilising ring to prevent
excessive "shaft wobble". Service and maintenance are affected by
the available space around the agitator, gearbox and seal assembly,
and on-site shaft installation and removal is limited by plant
height.
[0111] The protrusion of the seal and impeller shaft at tank bottom
restricts the placement of the sparger near the tank bottom. This
dimension affects the tank hydrodynamics and therefore its
amenability to change is important in specifying an optimal
design.
[0112] The downwards load of down pumping impellers together with
the liquid head have an accumulative greater load (compared to up
pumping or top-entry shaft) between the moving and stationary faces
of the seal resulting in greater wear of the seal faces.
Furthermore loss of over pressure in the condensate line supplying
the seal can result in the culture seeping into the seal. This
makes the subsurface seal a less sanitary design.
[0113] The submerged seal complicates the design of a free draining
bioreactor by compromising the position of the harvest drain valve.
Secondly the diameter of the harvest nozzle may be restricted thus
restricting the flow rate of harvest stream. Therefore a top entry
impeller shaft is used in the 20 000 litre bioreactor.
Baffles
[0114] The baffle requirement for centre mounted impeller is
critical to prevent vortex formation. The critical issues related
to baffles are baffle number, baffle width (W), baffle length
(H.sub.baffle) and baffle to tank wall clearance (W.sub.c).
[0115] The recommendation for four equally spaced baffles that are
0.1.times.T or 279 mm wide 1.1.times.H-H.sub.h or 3882 mm tall and
have a baffle to tank wall clearance, W.sub.c of 0.01.times.T or 28
mm.
[0116] The thickness of baffle is not specified but the thickness
needs to ensure rigidity to the radial component of the fluid flow.
Additionally thickness needs to ensure the baffle plates are not
warped during SIP thereby affecting the baffle to tank wall
clearance.
Impeller Type
[0117] High shear, such as Rushton (or Rushton-type), impellers
offer high power dissipation for gas dispersion but lack in axial
flow necessary for mixing and homogeneity. Additionally, agitation
from high shear impellers suffers from dangers of excessive cell
damage.
[0118] Table 4 shows the impellers tested at lab scale (12.2 litre)
that gave equivalent hydrodynamic and cell growth performance. The
hydrofoil is mounted above the high solidity pitched blade
impeller.
[0119] The Lightnin A310 and A315 at the D/T ratio described in
table 4 are used in the bioreactor.
TABLE-US-00004 TABLE 4 Impeller types short-listed for scale down
study Impellers D/T ratio .sup.1N.sub.p/.sup.2N.sub.q Vendor
Description A310 0.44 0.30/0.56 Lightnin Three bladed hydrofoil
design A315 0.46 0.75/0.73 Lightnin Four pitched- bladed high
solidity impeller SC-3 0.40 0.90/0.90 Chemineer Three bladed
hydrofoil design 3HS39 0.46 0.53/0.58 Philadelphia Four pitched-
Mixers bladed high solidity impeller .sup.1N.sub.p is
characteristic impeller power number. It is a measure of an
impeller efficiency to impart the kinetic energy of the rotating
impeller blades to the fluid. It is important in quantifying the
gas dispersion .sup.2N.sub.q is characteristic impeller flow
number. It is a measure of pumping ability of the impeller and is
important in quantifying fluid bulk movement.
Impeller to Tank Diameter, D/T Ratio
[0120] The diameter for axial flow impellers is recommended to be
less than 0.5.times.T. A diameter greater than this results in
disruption in axial flow, hence poor agitation and aeration.
[0121] Power dissipation into the bioreactor and Reynold's number
also need to be sufficiently high to maintain a turbulent (loaded)
regime. Therefore the selection of impeller diameter is a
compromise between choosing large enough diameter to ensure
adequate homogeneous mixing without exceeding the hydrodynamic
characteristics of the bioreactor. These include throttling axial
flow, insufficient power dissipation, exceeding upper limits of
impeller tip speed and creation of poorly mixed laminar zone.
[0122] Once a diameter is selected, than maintaining constant D/T
ratio is critical between scale down pilot vessels in order to
maintain the central assumption of scale studies--that of
maintaining geometric similarity.
[0123] The K.sub.La scale up correlation at 12.2 litre has been
determined for the four impellers at the D/T ratios shown in table
4. From a geometric similarity standpoint A310 diameter of 1.229 m
(D/T of 0.44) and A315 diameter of 1.285 m (D/T of 0.46) is
recommended. However a manway diameter can restrict the largest
impeller diameter that can be installed and removed to 1.219 m.
Therefore A310 and A315 to be 1.219 m diameter are used thereby
keeping with ease of impeller installation and removal and
maintaining close to the geometric similarity proposed in scale
down study.
The Impeller Clearance, D.sub.c Spacing, D.sub.s
[0124] The spacing between impellers in a bioreactor with multiple
impellers is an important dimension to consider. For a bioreactor
with dual Rushton turbine (radial flow) the ungassed power
consumption is equivalent to a single impeller when the dual
impeller are spaced less then 0.5.times.D along the shaft. At a
spacing of 2.times.D the power consumption becomes adductive. Thus
efficiency of the impeller is reduced when the impeller spacing
becomes less then 0.5.times.D and the requirement for multiple
impellers becomes unnecessary. It is important to note that
impeller spacing also impacts on the potential of creating dead
zones (poorly mixed zones) within the bioreactor. An additional
constraint on the choice of impeller spacing is discrete working
volumes required within the bioreactor.
[0125] The impeller spacing, D.sub.s, of 1.229.times.D.sub.bottom
(1498 mm) allows both impellers to remain submerged at the lowest
post-inoculation volume of 17392 litres with liquid head above the
upper impeller, D.sub.o, of 0.5.times.D.sub.top (615 mm) and off
bottom clearance, D.sub.C, of 0.75.times.D.sub.bottom (913 mm).
[0126] Table 5 highlights volumes that will form liquid surfaces or
lower liquid cover, above the impellers. Agitation needs to be
modified to avoid foaming at these critical volumes.
TABLE-US-00005 TABLE 5 Key operating volumes that cause interaction
with impellers and liquid surface Interaction Volume, L Potential
Operation Submerge top impeller with 17399 Minimum post inoculation
0.5D.sub.A310 liquid cover volume 17391 L Liquid surface touching
top 13973 Pre-inoculation volume of edge of top impeller 13913 L
liquid surface breakage Liquid surface touching 13283 Bolus
addition of pre-inoculation bottom edge of top impeller medium will
pass through this liquid head Submerge bottom impeller 8381 Bolus
addition of pre-inoculation with 0.5D.sub.A315 liquid cover medium
will pass through this liquid head Liquid surface touching top 5592
Bolus addition of pre-inoculation edge of bottom impeller medium
will pass through this liquid head Liquid surface touching 3291
Bolus addition of pre-inoculation bottom edge of bottom medium will
pass through this impeller liquid head .sup.(1)Minimum operating
volume with lower impeller submerged is 8379 litres and minimum
operating volume with both impellers submerged is 17399 litres
.sup.(2)The operating volume range is 13913 to 21739 litres.
Clearance of Top Impeller Below Liquid Surface, Do.
[0127] The breakage of the impeller blade above liquid surface is
undesirable as this will make the flow and power dissipation of the
impeller ineffective. In addition it will create unknown K.sub.La
values due to significant surface entrapment of headspace gas into
the fluid and excessive foam. D.sub.o is 0.3.times.D for radial
flow impellers and 0.5.times.D for axial flow impellers such as
A310. However as D.sub.o approaches 2.times.D the impeller provides
gentle blending duty. This is acceptable for the production
bioreactor application as K.sub.La study has shown that bioreactor
K.sub.La is influenced mostly by the bottom A315 impeller and the
top A310 impeller contributes to bulk mixing.
[0128] As a result of setting D.sub.c and D.sub.s at values D.sub.o
is maintained at an optimal range for the duration of operation of
the production bioreactor. During the course of a batch the liquid
cover above the top impeller will change from 0.5.times.D.sub.A310
and 1.08.times.D.sub.A310. The liquid cover above the top impeller
will increase as the bioreactor is fed nutrient feeds and alkali to
maintain constant pH. Table 6 shows a range of liquid cover above
the top impeller for a range of operating volumes.
TABLE-US-00006 TABLE 6 Key operating volumes and the liquid cover
above top impeller, Do Cylinderical height, H Do, mm Do as
Operating volume, L mm (inches) (inches) ratio of D.sub.A310
Pre-Harvest, 21739 L 3252 (128'') 1324 (52'') 1.08D.sub.A310
Pre-Harvest, 20000 L 2968 (117'') 1040 (41'') 0.85D.sub.A310
Post-Inoculation, 19231 L 2843 (112'') 915 (36'') 0.74D.sub.A310
Post-Inoculation, 17391 L 2543 (100'') 615 (24'') 0.5D.sub.A310
Pre-Inoculation, 15385 L 2215 (87'') 287 (11'') 0.23D.sub.A310
Pre-Inoculation, 13913 L 1973 (78'') 45 (2'') 0.04D.sub.A310
.sup.(1)Off bottom impeller clearance, Dc = 913 mm
(0.75D.sub.A315), Impeller separation, Ds = 1498 mm
(1.229D.sub.A315), tank ID of 2794 mm and height of ASME F&D
base plate, H.sub.h = 483 mm .sup.(2)D.sub.o = H - D.sub.s -
(D.sub.c - H.sub.h)
[0129] Agitation Rate--rpm, P/V and Tip Speed
[0130] Table 7 below specifies the agitation rate for the 20 000
litre bioreactor. The bioreactor is agitated typically at 20-260
W/m.sup.3, preferably at 55-85 100 W/m.sup.3. The agitation
strategy is being developed during the 500 litre pilot
fermentations. The agitation rate of 0 to 80.+-.1 rpm is therefore
used as an operational range.
TABLE-US-00007 TABLE 7 Agitation rate for the 20000 L bioreactor
Power per unit Agitation rate, rpm volume, W m.sup.-3 Tip Speed,
m/s Pre- Typically 28-30 can Typically 20 can be 1.8-1.9
inoculation be higher higher Post- Typically 56 can Typically 103,
can 3.6 can be inoculation be up to 80 be up to 260 up to 5.1 until
harvest
Mechanical Seals Specification
[0131] For bioreactor all seals are to be double mechanical seals
with a maximum "run out" or wobble tolerance of 0.2 mm. Three types
were considered; these include: [0132] Wet seal lubricated with
sterile condensate. [0133] Dry seal lubricated with sterile gas
such as N.sub.2 or CA. [0134] Non lubricated or floating seal that
are uni-rotational.
[0135] All mechanical seals are recommended to be serviced on an
annual basis. This requires the removal of seal from the bioreactor
and sending the seal assembly to the vendor. Therefore the design
must consider ease of routine maintenance.
[0136] The dry type seal (John Crane--5280D type) will produce 3 g
per year of shedding (seal face and seal seat material) composed of
resin impregnated carbon. This is based on continuous 24 hour
operation over a year. The amount of shedding for the wet seal is
significantly less. Therefore a wet condensate-lubricated seal is
adopted for all bioreactor double seals.
Bioreactor Aeration Requirement and Gassing Strategy
[0137] The aeration duty of the 20 000 litre bioreactor is governed
by: [0138] K.sub.La requirement. [0139] DOT control strategy.
[0140] pCO.sub.2 control/stripping strategy. [0141] Use of sintered
or fluted spargers.
[0142] The 20 000 litre bioreactor is designed to provide K.sub.La
values of up to 20 h.sup.-1 for processes with oxygen uptake rates
of 5 mmol.times.L.sup.-1.times.h.sup.-1. The bioreactor design
needs to be flexible enough to allow cultivation of processes
reaching 20.times.10.sup.6 cells.times.mL.sup.-1.
[0143] The aeration requirement can be achieved by a number of
different approaches. However the use of a fluted sparger with air
and oxygen enrichment to make up any deficit in oxygen transfer
rate (OTR) during peak oxygen demand was used. The advantages of
this approach are: [0144] Easier CIP and SIP validation of fluted
sparge design. [0145] Larger air throughput to aid dissolved
CO.sub.2 stripping. [0146] Reduced operating cost through the
avoidance of purchase of single use sintered elements.
[0147] The disadvantages of the approach selected above also need
to be considered. These include: [0148] Inherent lower K.sub.La for
the low power number impellers selected.
[0149] Therefore the bioreactor aeration design must have the
flexibility to be modified to meet the desired K.sub.La.
[0150] Table 8 describes the gassing requirements for the 20 000
litre bioreactor. The gas flow rates were scaled up on constant
superficial gas velocity.
[0151] Two spargers are used. The main or "DOT control" sparger
supplied by dual range clean air, mass flow controller (MFC) and
oxygen MFC with gas flow metered via a DOT control loop and a
CO.sub.2 MFC metering gas via the acid pH control loop. The dual
range MFC's are used to achieve precise flow control at the extreme
ends of the desired operating ranges.
[0152] The second or "ballast" sparger is supplied by a CA MFC to
which nitrogen is also supplied. It was measured that early DOT
control requires small nitrogen ballast to assist in early DOT
demand and lower the DOT to set point. The ballast sparger also
meters ballast air to facilitate stripping out excess
pCO.sub.2.
[0153] The headspace purge is used to allow removal of CO.sub.2 and
oxygen from the headspace. This is to facilitate better pH and
pCO.sub.2 control and dilution of high oxygen blend prior to
exhausting to environment. The ability to vary headspace flow rate
allows design of gassing strategy for various processes requiring
different blends of oxygen enrichment and control point
pCO.sub.2.
TABLE-US-00008 TABLE 8 Gas flow rate and MFC operating ranges for
the 20000 litre bio-reactor Operating Gas range Comments Head
Space.sup.1 1.) Clean air 1.) 0-1000 1.) Head space purging of SLPM
CO.sub.2 and O.sub.2 2.) Nitrogen 2.) Utility rated 2.) For rapid
DOT probe zeroing 3.) Helium 3.) Utility rated 3.) Tank integrity
testing DOT control Sparger 1.) Clean air.sup.2 1.) 10-500 1.) Gas
flow under DOT control SLPM 2.) Oxygen 2.) 10-100 2.) Gas flow
under DOT control Carbon dioxide.sup.3 SLPM 3.) 2-150 3.) Gas flow
under pH control SLPM Ballast Sparger 1.) Clean air 1.) 20-500 1.)
Variable ballast for SLPM dCO.sub.2 stripping 2.) Nitrogen.sup.4
2.) 20-500 2.) Early DOT control by SLPM variable flow .sup.1The
air and nitrogen gas flow into headspace enters via a bypass for
post SIP tank pressurisation. .sup.2Clean air gas flow operating
range achieved by a dual CA MFC at 5-50 SLPM and 50-500 SLPM
respectively. .sup.3CO.sub.2 gas flow operating range achieved by a
dual CO.sub.2 MFC at 2-30 SLPM and 30-150 SLPM respectively
.sup.4Both air and nitrogen gas flow metered from a common CA
MFC.
[0154] The bioreactor ports for sparger installation are designed
to fit pipe design of diameter of 51 mm. The position of port
should allow the placement of control sparger (D.sub.c-S.sub.c) at
a distance of 320 mm below the bottom edge of the lower impeller
and no greater 593 mm from tank bottom (S.sub.c).
[0155] This results in a S.sub.c value of 593 mm or
(0.65.times.D.sub.c) and this falls outside the acceptable range of
0.2.times.Dc to 0.6.times.Dc. However hydrodynamic trials in 500 l
suggest S.sub.c clearance of 0.41 to 0.71.times.D.sub.c has no
impact on measured K.sub.La.
[0156] A separate port for the installation of the ballast sparger
was also built. The position of this port allows the placement of
ballast sparger at a distance of 320 mm, (D.sub.c-S.sub.c) below
the bottom edge of the lower impeller and no greater then 593 mm
from tank bottom (S.sub.c). The requirement to add ballast from a
separate sparger is due to three reasons: [0157] Firstly, it
prevents dilution of oxygen or oxygen enriched DOT demand gas with
the ballast gas. This ensures the best OTR, as the oxygen
concentration gradient of the bubbles emerging from the sparger is
greatest. [0158] Secondly, it allows ballast sparger to be located
at a different position from DOT control sparger to avoid impacting
DOT control on delivering desired ballast for pCO.sub.2 control.
[0159] Thirdly, the ballast sparger can be independently designed
from the DOT control sparger.
[0160] The calculation of hole size and number of holes is iterated
until the target Reynold's number, Re of gas emerging from holes is
<2000 and the Sauter mean diameter for a bubble is 10-20 mm
during chain bubble regime. Table 9 shows the key specifications
for the control and ballast sparger for the 20 000 litre
bioreactor.
TABLE-US-00009 TABLE 9 Design specification for the 20 000 litre
bioreactor spargers DOT control Ballast Parameter sparger sparger
Gas flow, SLPM 850 500 Number of sparge holes 250 100 Orifice
diameter, d.sub.o, m 0.004 0.006 Gas flow, m.sup.3 s.sup.-1
1.42E-02 8.33E-03 Orifice area, m.sup.2 1.26E-05 2.83E-05 Total
orifice area, m.sup.2 3.14E-03 2.83E-03 Density of air, Kg m.sup.-3
1.166 1.166 Viscosity, Nm s.sup.-2 1.85E-05 1.85E-05 Sauter mean
diameter, d.sub.vs, 16.34 19.06 mm (d.sub.vs = 1.17 V.sub.o.sup.0.4
d.sub.o.sup.0.8 g.sup.-0.2) Gravitational acceleration, g m
s.sup.-2 9.807 9.807 Density difference, Kg m.sup.-3 1048.834
1048.834 Reynold's number, >2000 jetting regime 1139 1117 Gas
velocity at sparger, V.sub.o, m/s 4.51 2.95 Sparger length,
S.sub.L, m 1.077 1.077 Combined length to drill required holes, m
1.000 0.6 Number of rows to fit required holes 2 1 in length
S.sub.L Sparger to tank bottom clearance, Sc, m 0.593 0.593 Sparger
to bottom impeller clearance, Dc-Sc, m 0.320 0.320
[0161] A ring sparger of 0.8.times.D.sub.bottom (80% diameter of
bottom A-315 impeller diameter) is used to distribute the holes
under the blades and not the impeller hub. However the CIP and
installation of this configuration is difficult. Therefore
selection of sparger geometry that permits distribution of the
desired number of holes in a manner that is consistent with best to
distribute the holes and sanitary design can be used also.
[0162] As an option a crescent rather then straight pipe is
explored. The curvature of the crescent is 0.8.times.D.sub.bottom.
In order to aid installation and removal from side ports of the
bioreactor the crescent circumference is 240.degree. of the
complete circumference of 0.8.times.D.sub.bottom ring, this is 1077
mm.
[0163] The DOT control sparger is 1077 mm long and has a 51 mm
diameter. The holes have a 4 mm diameter. A total of 250 holes
divided into 2 rows (2.times.125) at 45.degree. from the dorsal
(vertical) are used. Drain holes of 4 mm diameter on both ends of
the sparger are drilled on the ventral side of the sparger to aid
free CIP drainage of the sparger.
[0164] The ballast sparger is 1077 mm long and of 51 mm diameter
and has a total of 100 6 mm diameter holes in a single dorsal row.
Drain holes of 4 mm diameter on both ends of the sparger are
drilled on the ventral side of the sparger to aid free CIP drainage
of the sparger.
Position of Probes, Addition and Sample Ports
[0165] The probe ring position must be placed in a well-mixed
representative region of the bioreactor. Additional considerations
included working volume range and ergonomic operations. The
location of probe ports, sample valve and addition points were
considered together to avoid transitory spikes. Furthermore the
position of the sample valve with respect to controlling probes
needs to permit accurate estimation of off-line verification of the
measured process parameter. This is shown in table 10.
TABLE-US-00010 TABLE 10 Probe, addition and sampling port
specification for the 20000 litre bioreactor .sup.2Diameter, mm
.sup.1Position, mm Probe/Port Location (inches) (inches) Rational
Temperature (main) Lower ring 38.1 (1.5'') 1.) 913 (36'') In the
plane of 2.) 30.degree. centre-line of bottom impeller Temperature
Lower ring 38.1 (1.5'') 1.) 913 (36'') In the plane of (backup) 2.)
170.degree. centre-line of bottom impeller pH (main) Lower ring
38.1 (1.5'') 1.) 913 (36'') In the plane of 2.) 10.degree.
centre-line of bottom impeller pH (backup) Lower ring 38.1 (1.5'')
1.) 913 (36'') In the plane of 2.) 20.degree. centre-line of bottom
impeller DOT (main) Lower ring 25.0 (0.98'') 1.) 913 (36'') In the
plane of 2.) 150.degree. centre-line of bottom impeller DOT
(backup) Lower ring 25.0 (0.98'') 1.) 913 (36'') In the plane of
2.) 160.degree. centre-line of bottom impeller pCO.sub.2 (spare)
Lower ring 50.8 (2'')tbd 1.) 913 (36'') In the plane of 2.)
20.degree. centre-line of bottom impeller Biomass (spare) Lower
ring 50.8 (2'') 1.) 913 (36'') In the plane of 2.) 160.degree.
centre-line of bottom impeller Spare probe port Lower ring 25.0
(0.98'') 1.) 913 (36'') In the plane of (DOT-type) 2.) 150.degree.
centre-line of bottom impeller Spare probe port Lower ring 38.1
(1.5'') 1.) 913 (36'') In the plane of (pH-type) 2.) 10.degree.
centre-line of bottom impeller Sample valve (main) Lower ring 12.7
(0.5'') 1.) 913 (36'') NovAseptic 2.) 40.degree. type Sample valve
Lower ring 12.7 (0.5'') 1.) 913 (36'') NovAseptic (backup) 2.)
50.degree. type Alkali addition 1 - Lower ring 50.8 (2'') 1.) 913
(36'') Diametrically Tank 1 2.) 190.degree. opposite pH probes
Alkali addition 2 - Centre-line 50.8 (2'') 1.) 2411 (95'')
Diametrically Tank 1 of upper 2.) 190.degree. opposite pH impeller
probes Continuous feed 1 - Lower ring 50.8 (2'') 1.) 913 (36'')
Diametrically Tank 2 2.) 200.degree. opposite pH probes Continuous
feed 2 - Lower ring 50.8 (2'') 1.) 913 (36'') Diametrically Tank 3
2.) 210.degree. opposite pH probes DOT control sparger N/A 101.6
(4'') 1.) 593 (23'') Diametrically orifice 2.) 0.degree. opposite
ballast sparger Ballast sparger orifice N/A 101.6 (4'') 1.) 593
(23'') Diametrically 2.) 180.degree. opposite control sparger
Overlay gas Head plate 101.6 (4'') 1.) N/A Diametrically 2.)
135.degree. opposite vent out Exhaust vent out Impeller 50.8 (2'')
1.) N/A Diametrically flange 2.) 315.degree. opposite over- plate
lay gas in Harvest valve Base plate 76.2 (3.0'') 1.) N/A NovAseptic
2.) Centre type to allow free draining Antifoam addition Head plate
50.8 (2'') 1.) N/A Liquid surface/ 2.) 170.degree. 0.25T from tank
centre Shot feed 1 - LS1 Head plate 50.8 (2'') 1.) N/A Liquid
surface 2.) 190.degree. Shot feed 2 - Glucose Head plate 50.8 (2'')
1.) N/A Liquid surface shot 2.) 180.degree. Small add - Spare Head
plate 50.8 (2'') 1.) N/A Liquid surface - 2.) 200.degree. directed
into vessel wall Media inlet Head plate 101.6 (4'') 1.) N/A Nozzle
directed 2.) 310.degree. onto vessel wall Inoculum transfer Head
plate 101.6 (4'') 3.) N/A Nozzle directed from 4000 L to 4.)
320.degree. onto 20000 L vessel wall CIP - Spray ball Impeller 76.2
(3'') 1.) N/A CIP'ing of flange 2.) 270.degree. highest point plate
CIP - Spray ball Head plate 76.2 (3'') 5.) N/A As per CIP 6.)
60.degree. design CIP - Spray ball Head plate 76.2 (3'') 1.) N/A As
per CIP 2.) 180.degree. design CIP - Spray ball Head plate 76.2
(3'') 1.) N/A As per CIP 2.) 300.degree. design Pressure indicating
Head plate 38.1 (1.5'') 1.) N/A As per vessel transmitter (PIT) 2.)
60.degree. vendor design Pressure gauge Head plate 38.1 (1.5'') 1.)
N/A As per vessel 2.) 50.degree. vendor design Rupture disc Head
plate 101.6 (4'') 1.) N/A As per vessel 2.) 280.degree. vendor
design Spare nozzle Head plate 101.6 (4'') 1.) N/A As per vessel
2.) 160.degree. vendor design Sight glass Head plate 101.6 (4'')
1.) N/A As per vessel 2.) 70.degree. vendor design Light glass Head
plate 76.2 (3'') 1.) N/A As per vessel 2.) 75.degree. vendor design
Manway Head plate 457.2 (18'') 1.) N/A Personnel 2.) 90.degree.
entry Agitator head/flange Head plate 1320.8 (52'') N/A
Entry/removal Impeller shaft and Agitator 304.8 (12'') N/A
Entry/removal seal manway head/ flange .sup.1Measured from the
tangential line of the base plate. Degrees pertain to plane of
clockwise rotation. .sup.2Diameter of nozzle at bioreactor.
Addition Ports, Surface and Subsurface
[0166] The need to determine addition ports that terminate at
liquid surface and those that are subsurface was determined by
operational scenarios and the effects of feed strategy on process
control.
[0167] Currently the protein free process has two continuous feeds
that need to be discharged in well-mixed area of the bioreactor.
Additional provision for glucose and an "LS1-type" shot addition is
also integrated in the well mixed region. The foam is controlled by
surface addition of 1 in 10 diluted C-emulsion. The inoculation of
seed into the pre-inoculation bioreactor is served by avoiding
build up of foam which will arise as the culture is dropped onto
the surface of the medium. Following ports were designed: [0168]
Six surface additions with media inlet, inoculum inlet, one small
addition inlet directed into the wall of the vessel while the
others dropped onto the liquid surface away from the tank wall.
[0169] Four subsurface additions comprised of inlets from the two
feed tanks and bi-level inlet from the alkali tank.
Sample Ports
[0170] The sample port design allows a representative sample to be
taken from the bioreactor. Therefore any residual material must be
as small as possible. The samples taken are used to determine off
line checks for dissolved gases, pH, nutrients and biomass
concentration. The orifice of the port opening is large enough to
prevent sieving causing biomass aggregates to be retained. The 2 mm
orifice NovaSeptum sampling device was used. However this has to be
balanced with the desire to keep residual volume of the port low.
The port needs to be positioned in a well-mixed zone adjacent to
the probes that need to be verified by off-line checks and will be
determined via nozzle position (see table 10).
Add Tanks
[0171] In order to reduce cost and time the add-tanks supplying the
bioreactor are of modular design. The production bioreactor has
three 2500 litre nominal volumes add tanks. The add tanks are
filled at 25 l/min. The flow rate of feeds from the add tanks to
the bioreactor is controlled at 0.2 to 1.0 millilitres of feed per
litre of post-inoculation bioreactor volume per hour (ml/l/h). It
is expected that feed rate is controlled at .+-.5% of set
point.
[0172] The production bioreactor is serviced by three 1372 mm ID by
1880 mm add tanks. These tanks have the capability to be cleaned
and sterilised independently and together with the production
bioreactor.
Manway
[0173] Access into the bioreactor is required for certain service
operations. Access can be gained by considering a flanged head
plate or incorporation of a manway into the head plate. The need
for access into the bioreactor is for: [0174] Installation of
impellers. [0175] Installation and replacing of impeller and
impeller shaft. [0176] Installation and replacing of mechanical
seal. [0177] Service of vessel furniture. [0178] Potential
modification of sparger position to obtain desired hydrodynamic
characteristics.
[0179] The size of the manway must be sufficient to allow access
for the above objectives. The manway used was of sufficient
diameter to allow the removal of two impellers of 1219 mm
diameter.
Volume Measurement
[0180] The design ensures that any sensor gives sufficient
precision in volume measurement around the operating range.
[0181] The volume measurement in bioreactor is able to measure a
range of 13 000 to 25 000 litres. The sensor sensitivity needs to
be at least 0.5% of full span.
[0182] Volume measurement in the feed add-tanks and alkali tank is
able to measure 0 to 2200 and 2500 litres respectively. The sensor
sensitivity needs to be at least 0.2% of full span. This will
permit hourly verification of feed flow rate at the minimum flow
rate of 0.2 ml/l per hour or 3.51 per hour by measuring the volume
decrease in the add tanks.
Bioreactor Temperature Control
[0183] The medium is brought to operating temperature and pH by
process control. This is achieved by "gentle" heating of the jacket
(avoid high temperature at vessel wall). The temperature control
range during operation is 36 to 38.degree. C. with an accuracy of
.+-.0.2.degree. C. at set point.
Jacket
[0184] The bioreactor jacket area is specified with the following
considerations in mind:-- [0185] Steam sterilisation at
121-125.degree. C. [0186] Warming up of medium from 10.degree. C.
to 36.5.degree. C. in <2 h. [0187] All points within the
bioreactor must reach .+-.0.2.degree. C. of set point, typically
36.5.degree. C., as measured by thermocouples. [0188] Chilling of
medium from 36.5.degree. C. to 10.degree. C. in <2 h.
Bioreactor pH Control
[0189] The process pH is monitored and controlled with probes
connected via a transmitter to a DCS based process controller. The
process is be controlled by addition of CO.sub.2 to bring the pH
down to set point and addition of alkali to bring pH up to set
point. pH is controlled at .+-.0.03 of set point.
[0190] Alkali is added through two addition points to distribute
the alkali. This ensures quicker blending of alkali in the event of
long re-circulation time in the tank. The CO.sub.2 is added via the
control sparger.
[0191] Control and back-up probes are located in the lower port
ring at 913 mm (see table 10) from tank bottom. Additionally the pH
probes are located diametrically opposite the alkali addition
points into the bioreactor.
Bioreactor DOT Control
[0192] Dissolved oxygen is monitored and controlled with
polarographic DOT probe. The DOT set point maintained by sparging:
[0193] Initial N.sub.2 ballast and/or air on demand. [0194] Air
ballast with air on demand. [0195] Air ballast with oxygen on
demand. [0196] Reversing gas usage once oxygen demand
decreases.
[0197] Cascade DOT control allows DOT set point to be maintained
through changes in the ballast and demand gas in conjunction with
ramping of agitator speed.
[0198] In order to control pCO.sub.2 the ballast required to strip
out excess dCO.sub.2 impacts DOT control. Therefore the DOT control
is considered together with pCO.sub.2 control for those processes
where metabolic CO.sub.2 is liberated. DOT is controlled at .+-.2%
of set point. Control and back-up probes are located in the lower
port ring at 913 mm from tank bottom.
Bioreactor Dissolved CO.sub.2 Control
[0199] The process dCO.sub.2 is monitored with an pCO.sub.2 probe
and excess dCO.sub.2 is stripped by gassing CA through the ballast
sparger. The optimal position for this probe is close to the pH
probes.
Feed Addition Control
[0200] The feeds (SF22 and amino acid) are high in pH and
osmolality. Therefore bolus additions need to be avoided to
maintain good pH control. However the control of desired flow rates
(.+-.5% of set point) is technically challenging. Therefore an
addition strategy that encompasses point of addition with delivery
mode avoids the circulation of feed bolus and potential variations
of pH control.
[0201] Therefore the point of addition is in the plane of the
centre-line of the bottom impeller that is 913 mm from tank bottom
to assist in the rapid blending of feed bolus.
Antifoam Addition Control
[0202] Antifoam (C-emulsion) addition is added as required to
maintain the bioreactor liquid surface free of foam. A working
stock of 1 in 10 diluted C-emulsion can be dosed on the liquid
surface. The antifoam suspension is continuously agitated in the
storage container to prevent partitioning. It is important to dose
the antifoam close to the centre of the tank to diminish the
effects of the radial component of the fluid flow carrying the
antifoam to the tank walls where it will adhere. Therefore the
addition point is 0.25.times.T toward the tank centre or 699 mm
from tank centre.
EXAMPLE 2
4000 litre Bioreactor
Vessel Geometry
[0203] The vessel geometry for the 4000 litre bioreactor was
determined by an iterative design basis in which the maximum
working volume, freeboard straight side distance aspect ratio
(H.sub.L/T) and impeller to tank diameter ratio (D/T) are altered
until an acceptable aspect ratio is achieved.
Bioreactor Aspect Ratio H.sub.L/T
[0204] Table 11 describes the aspect ratios in the 4000 litre
bioreactor at the various operating volumes during normal
processing. These aspect ratios arise from the selection of tank ID
and the operating volume required. From a processing perspective
the mixing requirements at the three operating conditions are
different. During pre-inoculation stage the bioreactor mixing is
important to allow medium to equilibrate with minimal K.sub.La
requirement. However for post-inoculation and pre-transfer stages
both mixing and K.sub.La are important considerations. Therefore
both these features were tested at the aspect ratio range.
TABLE-US-00011 TABLE 11 Key operating volumes and aspect ratios in
the 4000 litre bioreactor Volume, L Liquid head, mm Aspect ratio,
H.sub.L/T Pre-Inoculation 1.) 1914 1.) 1031 1.) 0.63 2.) 2782 2.)
1448 2.) 0.89 3.) 3077 3.) 1590 3.) 0.98 Post Inoculation & 1.)
2153 1.) 1146 1.) 0.70 Pre-transfer 2.) 3478 2.) 1783 2.) 1.10 3.)
3846 3.) 1960 3.) 1.21
Tank Diameter
[0205] The tank diameter was altered to obtain the optimal aspect
ratio H.sub.L/T. Changes to tank internal diameter are limited by
acceptable aspect ratio and plant footprint. The tank ID is 1626
mm.
Tank Height
[0206] Tank height is determined from the maximum operating volume,
aspect ratio H.sub.L/T, freeboard straight side length, base and
top plate design. The final tank height is a compromise value
determined from volumetric contingency for foam, plant height and
acceptable impeller shaft length. The head to base tan line height
is 2817 mm.
Freeboard Height
[0207] The freeboard height of 500 mm (1039 litre or 27% v/v of the
maximum operating volume) is used for this seed bioreactor.
Head and Base Plate
[0208] The base and head plate design is a ASME F&D design for
this seed bioreactor.
Bioreactor Agitation Requirement
[0209] The agitation of the bioreactor is to achieve rapid mixing,
maintain homogeneity, maintain mammalian cells in suspension and
gas bubble dispersion. The underlying issue for achieving the above
objectives is minimising cell damage through shear forces
originating from impeller geometry and eddies or vortices created
behind the impeller blades. A compromise of the above objectives
was achieved by selection of an appropriate impeller type.
Bottom Versus Top Driven Impeller Shaft
[0210] The motor drive is top mounted for the benefits already
highlighted.
Baffles
[0211] The baffle requirement for centre mounted impeller is
critical to prevent vortex formation. The critical issues related
to baffles are baffle number, baffle width (W), baffle length
(H.sub.baffle) and baffle to tank wall clearance (W.sub.c).
[0212] Four equally spaced baffles that are 0.1.times.T or 163 mm
wide 1.1.times.H-H.sub.h or 2195 mm tall and have a baffle to tank
wall clearance, W.sub.c of 0.01.times.T or 16 mm were used.
[0213] The thickness of the baffles is not specified but the
thickness needs to ensure rigidity to the radial component of the
fluid flow. Additionally thickness needs to ensure the baffle
plates are not warped during SIP thereby affecting the baffle to
tank wall clearance.
Impeller Type, Size and Number
[0214] The impellers for this bioreactor are identically formed to
the 20000 litre vessel and have a identical D/T ratio of 0.44. The
bottom impeller is a Lightnin's A315 at 710 mm of diameter and the
top impeller is a Lightnin's A310 at 710 mm of diameter.
The Impeller Spacing, D.sub.c, D.sub.s and D.sub.o
[0215] The impeller spacing, D.sub.s, between the centre-line of
the top impeller and the centre-line of the lower impeller is
1.229.times.D.sub.bottom or 872 mm. The off bottom impeller
clearance, D.sub.c is 0.75.times.D.sub.bottom or 531 mm. This
allows the lower impeller to remain submerged at the lowest
post-inoculation volume of 2153 litres and both impellers submerged
at 3367 litres with liquid head above the upper impeller (D.sub.o)
of 0.5.times.D.sub.top or 358 mm.
[0216] Table 12 highlights the volumes that will form liquid
surfaces or lower liquid cover above the impeller. Agitation can be
modified to avoid foaming at these critical volumes.
TABLE-US-00012 TABLE 12 Key operating volumes that cause
interaction with impellers and liquid surface Interaction Volume, L
Potential Operation Submerge top impeller 3433 Volume seen during
inoculation with 0.5D.sub.A310 liquid of 1 in 5 processes cover
Liquid surface touching 2758 Volumes seen during top edge of top
impeller pre-inoculation fill of 1 in 5 processes. Liquid surface
touching 2621 Volumes seen during bottom edge of top impeller
pre-inoculation fill of 1 in 5 processes. Submerge bottom impeller
1654 Volumes seen during with 0.5D.sub.A315 liquid pre-inoculation
fill of 1 cover in 5 processes. Liquid surface touching 1104
Volumes seen during top edge of bottom impeller pre-inoculation
fill of 1 in 5 and 1 in 9 processes. Liquid surface touching 650
Volumes seen during bottom edge of bottom pre-inoculation fill of 1
impeller in 5 and 1 in 9 processes. .sup.(1)Minimum operating
volume with lower impeller submerged is 1654 litres and minimum
operating volume with both impellers submerged is 3433 litres
.sup.(2)The operating volume range is 1914 to 3846 litres.
[0217] The 4000 l bioreactor can operate at two discrete
post-inoculation volumes with either the lower impeller submerged
(during cultivation of 1 in 9 seeding process) or with both
impellers submerged (during 1 in 5 seeding process), table 13 shows
the liquid cover obtained for the upper and lower impeller during
its operation.
[0218] A liquid cover of 0.67 to 0.82.times.D.sub.bottom above the
lower A315 impeller is observed during cultivation of the 1 in 9
seeded processes. This is within the recommendations of 0.5 to
1.times.D.
[0219] A liquid cover of 0.06 to 0.78.times.D.sub.top above the top
A310 impeller is observed during cultivation of 1 in 5 seeded
process. The lower liquid cover is outside the recommendation.
However this liquid cover is observed during pre-inoculation when
mixing and agitation are less critical.
TABLE-US-00013 TABLE 13 Key operating volumes and the liquid cover
above top impeller, Do and bottom impeller, D.sub.Bo Cylinderical
Do, D.sub.Bo, Do as ratio D.sub.Bo as ratio Operating volume, L
height, H (mm) mm mm of D.sub.A310 of D.sub.A315 Post-Inoculation
and 863 or 34'' -- 614 or 24'' -- 0.82D.sub.A315 Pre-Transfer, 2153
L Post-Inoculation and 1501 or 59'' 380 or 15'' -- 0.53D.sub.A310
-- Pre-Transfer, 3478 L Post-Inoculation and 1678 or 66'' 557 or
22'' -- 0.78D.sub.A310 -- Pre-Transfer, 3846 L Pre-Inoculation,
1914 L 748 or 29'' -- 499 or 20'' -- 0.67D.sub.A315
Pre-Inoculation, 2782 L 1166 or 46'' 45 or 2'' -- 0.06D.sub.A310 --
Pre-Inoculation, 3077 L 1308 or 52'' 187 or 7'' -- 0.26D.sub.A310
-- .sup.(1)Off bottom impeller clearance, Dc = 531 mm
(0.75D.sub.A315), Impeller separation, Ds = 872 mm
(1.229D.sub.A315), tank ID of 1626 mm and Height of ASME F&D
base plate, H.sub.h = 282 mm .sup.(2)Do = H - Ds - (Dc - H.sub.h)
and D.sub.Bo = H - (Dc - H.sub.h)
Agitation Rate--rpm, P/V and Tip Speed
[0220] Table 14 specifies the agitation rate for the 4000 litre
bioreactor. The bioreactor will be agitated typically at 20-260
W/m.sup.-3, preferably at 55-85 W/m.sup.-3. The agitation strategy
was developed during the 500 litre pilot fermentations. An
agitation rate of 0 to 88.+-.1 rpm is therefore used as an
operational range.
TABLE-US-00014 TABLE 14 Agitation rate for the 4000 L bioreactor
Agitation rate, rpm Power per unit volume, W/m3 Tip Speed, m/s
.sup.10-88 0-150 0.0-3.3 .sup.20-86 0-150 0.0-3.2 .sup.1When both
impellers submerged .sup.2When bottom impeller submerged
Mechanical Seals Specification
[0221] A double mechanical seal that is condensate lubricated is
used as described.
Bioreactor Aeration Requirement
[0222] Table 15 shows the gas flow, based upon scale up of constant
superficial gas velocity, for DOT and pH control during the
inoculum expansion in the 4000 litre bioreactor. Oxygen is not
required for DOT control. However oxygen enriched air can be used
to facilitate lower gassing to prevent excess foaming. It is
recommended that a smaller range N.sub.2 MFC should supply nitrogen
for early DOT control and reducing deviant, high levels of DOT.
TABLE-US-00015 TABLE 15 Gas flow rate and MFC operating ranges for
the 4000 litre bioreactor Gas Operating range Comments Head
Space.sup.1 1. Clean air 1. 0-200SLPM 1. Head space purging of
CO.sub.2 and 2. Nitrogen 2. Utility rated O.sub.2 3. Helium 3.
Utility rated 2. For rapid DOT probe zeroing 3. Tank integrity
testing Control Sparger 1. Clean air.sup.1 1. 10-60SLPM 1. Gas flow
under DOT control 2. Oxygen 2. 1.0-10SLPM 2. Gas flow under DOT
control 3. Carbon dioxide 3. 1.0-20SLPM 3. Gas flow under pH
control 4. Nitrogen.sup.2 4. 2.0-15SLPM 4. Early DOT control by
ballast 5. Helium 5. Utility rated 5. Tank integrity testing
.sup.1The air and nitrogen gas flow into bioreactor via a bypass
for post SIP tank pressurisation. .sup.2Nitrogen delivered via the
2 to 15SLPM N.sub.2 MFC and could be used during early DOT
control
[0223] The calculation of hole size and number of holes, for the
fluted sparger, is iterated until the target Reynolds number of gas
emerging from holes is <2000 and the Sauter mean bubble diameter
for a bubble chain regime is approximately 10 mm.
[0224] Table 16 show the key sparger design specification for the
4000 litre bioreactor. The sparger length, S.sub.L of 568 mm is
determined for pipe geometry. The holes are distributed on either
end of the sparger to prevent bubble liberating directly under the
A315 hub. Alternatively a crescent geometry can be used. The pipe
diameter is selected to aid spacing of the desired number of holes.
The diameter is 38 mm. The 100 2 mm holes are located on the dorsal
surface of the sparger with a single 2 mm hole located on the
ventral surface to aid free CIP drainage of the sparger.
[0225] The bioreactor port for sparger installation is designed to
a fit pipe design of diameter of 38 mm. The position of the port
allows the placement of a control sparger at a distance of 194 mm,
D.sub.c-S.sub.c below the bottom edge of the lower impeller and no
greater 337 mm from tank bottom, S.sub.c.
TABLE-US-00016 TABLE 16 Design specification for the 4000 litre
bioreactor sparger Parameter Control Sparger Gas flow, SLPM 105
Number of sparge holes 100 Orifice diameter, d.sub.o, m 0.002 Gas
flow, m.sup.3 s.sup.-1 1.75E-03 Orifice area, m.sup.2 3.14E-06
Total orifice area, m.sup.2 3.14E-04 Density of air, Kg m.sup.-3
1.166 Viscosity, Nm s.sup.-2 1.85E-05 Sauter mean diameter, mm
(d.sub.vs = 1.17 V.sub.o.sup.0.4d.sub.o.sup.0.8g.sup.-0.2) 10.21
Gravitional acceleration, g, m s.sup.-2 9.807 Density difference,
Kg m.sup.-3 1048.834 Reynold's number, >2000 jetting regime 704
Gas velocity at sparger, V.sub.o, m/s 5.57 Sparger length, S.sub.L,
m 0.568 Combined length to drill required holes, m 0.2 Number of
rows to fit required holes in length S.sub.L 1 Sparger to tank
bottom clearance, Sc, m 0.337 (13'') Sparger to bottom impeller
clearance, Dc-Sc, m 0.194 (8'')
Position of Probes, Addition and Sample Ports
[0226] The design basis for positioning of probes, addition and
sample ports has been covered in example 1 and are listed in table
17:
TABLE-US-00017 TABLE 17 Probe, addition and sampling port
specification for the 4000 litre bioreactor .sup.2Diameter,
.sup.1Position, Probe/Port Location mm (inches) mm (inches)
Rational Temperature (main) Lower ring 38.1 (1.5'') 1.) 531 (21'')
In the plane of 2.) 30.degree. centre-line of bottom impeller
Temperature Lower ring 38.1 (1.5'') 1.) 531 (21'') In the plane of
(backup) 2.) 170.degree. centre-line of bottom impeller pH (main)
Lower ring 38.1 (1.5'') 1.) 531 (21'') In the plane of 2.)
10.degree. centre-line of bottom impeller pH (backup) Lower ring
38.1 (1.5'') 1.) 531 (21'') In the plane of 2.) 20.degree.
centre-line of bottom impeller DOT (main) Lower ring 25.0 (0.98'')
1.) 531 (21'') In the plane of 2.) 150.degree. centre-line of
bottom impeller DOT (backup) Lower ring 25.0 (0.98'') 1.) 531
(21'') In the plane of 2.) 160.degree. centre-line of bottom
impeller Spare-1 (nutrient) Lower ring 25.0 (0.98'') 1.) 531 (21'')
In the plane of 2.) 170.degree. centre-line of bottom impeller
Spare-2 (pCO.sub.2) Lower ring 38.1 (1.5'') 1.) 531 (21'') In the
plane of 2.) 180.degree. centre-line of bottom impeller Spare-3
(biomass) Lower ring 50.8 (2'') 1.) 531 (21'') In the plane of 2.)
190.degree. centre-line of bottom impeller Sample valve Lower ring
12.7 (0.5'') 1.) 531 (21'') NovAseptic type (main) 2.) 40.degree.
Alkali addition Lower ring 50.8 (2'') 1.) 531 (21'') Diametrically
2.) 190.degree. opposite pH probes Feed 1 Lower ring 50.8 (2'') 1.)
531 (21'') Diametrically 2.) 200.degree. opposite pH probes Feed 2
Lower ring 50.8 (2'') 1.) 531 (21'') Diametrically 2.) 210.degree.
opposite pH probes Antifoam addition Head plate 50.8 (2'') 1.) N/A
Liquid surface/ 2.) 170.degree. 0.25T from tank centre Spare
surface addition Head plate 50.8 (2'') 3.) N/A Liquid surface 4.)
180.degree. directed to vessel wall DOT control sparger N/A 50.8
(2'') 337 (13'') orifice 1.) 0.degree. Overlay gas Head plate 101.6
(4'') 1.) N/A Diametrically 2.) 135.degree. opposite vent out
Exhaust vent out Head plate 50.8 (2'') 1.) N/A Diametrically 2.)
315.degree. opposite overlay gas in Transfer valve Base plate 76.2
(3.0'') 1.) N/A NovAseptic type 2.) Centre to allow free draining
Inoculum transfer Head plate 101.6 (4'') 1.) N/A Directed into from
1000 L to 4000 L 2.) 320.degree. vessel wall Media inlet Head plate
101.6 (4'') 3.) N/A Directed into 4.) 310.degree. vessel wall CIP -
Spray ball Impeller 76.2 (3'') 1.) N/A CIP'ing of highest flange
plate 2.) 270.degree. point CIP - Spray ball Head plate 76.2 (3'')
1.) N/A 2.) 60.degree. CIP - Spray ball Head plate 76.2 (3'') 1.)
N/A 2.) 180.degree. CIP - Spray ball Head plate 76.2 (3'') 1.) N/A
2.) 300.degree. Pressure indicating Head plate 38.1 (1.5'') 1.) N/A
transmitter (PIT) 2.) 60.degree. Pressure gauge Head plate 38.1
(1.5'') 1.) N/A 2.) 50.degree. Rupture disc Head plate 101.6 (4'')
1.) N/A 2.) 280.degree. Spare nozzle Head plate 101.6 (4'') 1.) N/A
2.) 160.degree. Sight glass Head plate 101.6 (4'') 1.) N/A 2.)
70.degree. Light glass Head plate 76.2 (3'') 1.) N/A 2.) 75.degree.
Agitator head/flange Head plate 813 (32'') N/A Entry/removal
Impeller shaft and Agitator 152 (6'') N/A Entry/removal seal manway
head/ flange .sup.1Measured from the tangential line of the base
plate. Degrees pertain to plane of clockwise rotation.
.sup.2Diameter of nozzle at bioreactor.
Addition Ports, Surface and Sub-Surface
[0227] The need to categorise additions ports that terminate at
liquid surface and those that are subsurface is determined by the
operational scenarios and effects of feed strategy on process
control.
[0228] The 4000 litre bioreactor has been designed to accept two
subsurface feeds and alkali that need to be discharged in
well-mixed area of the bioreactor. The foam is controlled by
surface addition of 1 in 10 diluted C-emulsion. A single spare
above surface addition port directed to the vessel wall is also
designed for future flexibility. The splashing of culture onto the
surface of the medium during inoculation of the seed bioreactor can
be avoided to prevent build up of foam. Therefore the inoculum
addition port is above surface and directed to the vessel wall. The
use of the harvest port in the base plate is the ideal port for
removal of inoculum during transfer of inoculum. Additionally the
medium addition port is directed to the vessel wall. In summary the
total addition ports are: [0229] Four surface additions with medium
inlet, inoculum inlet and a spare small addition directed to the
vessel wall and addition port for antifoam dropped on to the liquid
surface away from the vessel wall. [0230] Three subsurface
additions for feeds and alkali.
Sample Ports
[0231] The sample port design is similar to that specified for the
20 000 litre bioreactor.
Volume Measurement
[0232] The level sensor is able to measure up to 4000 litres with
an accuracy.+-.0.5% of full span.
Bioreactor Temperature Control
[0233] The 1914 to 3077 litre of medium are brought to operating
temperature, typically 36.5.degree. C. by process control. This is
achieved by "gentle" heating of the jacket and avoid high
temperature at vessel wall.
Jacket
[0234] The bioreactor jacket area is specified with the following
considerations in mind: [0235] Steam sterilisation at
121-125.degree. C. [0236] Warming up of 1914-3077 litres of medium
from 10.degree. C. to 36.5.degree. C. in <2 h. [0237] All points
within the bioreactor must reach .+-.0.2.degree. C. of set point,
typically 36.5.degree. C. as measured by thermocouples. [0238]
Chilling of 1914-3077 litres of medium from 36.+-.2.degree. C. to
10.degree. C. in 2 h.
Bioreactor pH Control
[0239] The process pH is monitored and controlled with probes
connected via a transmitter to a DCS based process controller. The
process pH is controlled by addition of CO.sub.2 through the
control sparger to bring the pH down to set point and addition of
alkali to bring pH up to set point.
[0240] Alkali is added through at least one subsurface port at
centre-line of the bottom impeller. The CO.sub.2 will be added via
the control sparger.
[0241] Control and backup probes are in the lower port ring at 531
mm from tank bottom as shown in table 17.
Bioreactor DOT control
[0242] Dissolved oxygen is monitored and controlled with
polarographic DOT probe. The DOT set point maintained by sparging:
[0243] Initial N.sub.2 ballast and/or air on demand. [0244] Air
ballast with air on demand. [0245] Air ballast with oxygen on
demand.
[0246] DOT control allows DOT set point to be maintained through
interchangeable use of oxygen or air as demand gas. It is not
envisaged that pCO.sub.2 control is required in the inoculum
bioreactor. Control and backup probes are in the lower port ring at
531 mm from tank bottom as shown in table 17.
Feed Addition Control
[0247] The point of addition is 531 mm from tank bottom, in the
plane of the centre-line of the lower impeller to assist in the
rapid dissipation of feed bolus.
Antifoam Addition Control
[0248] The addition point is at surface projecting 0.25.times.T
toward the tank centre or 407 mm from centre of tank.
EXAMPLE 3
1000 Litre Bioreactor Specification
Vessel Geometry
[0249] The vessel geometry for the 1000 litre bioreactor was
determined by an iterative design basis in which the maximum
working volume, freeboard straight side distance, aspect ratio
(H.sub.L/T) and impeller to tank diameter ratio (D/T) are altered
until an acceptable aspect ratio is achieved.
Bioreactor Aspect Ratio H.sub.L/T
[0250] Table 18 below describes the aspect ratios in the 1000 litre
bioreactor at various operating volumes during normal processing.
These aspect ratios arise from the selection of tank ID and the
operating volume required. From a processing perspective the mixing
requirements at the different operating conditions are different.
During pre-inoculation stage the bioreactor mixing is important to
allow medium to equilibrate with minimal K.sub.La requirement.
However with post-inoculation and pre-transfer stages both mixing
and K.sub.La are important considerations. Therefore both of these
features were tested at the aspect ratio range.
TABLE-US-00018 TABLE 18 Key operating volumes and aspect ratios in
the 1000 litre bioreactor Aspect ratio, Volume, L Liquid head, mm
H.sub.L/T Stage N-3 250 484 0.56 Pre-Inoculation Stage N-3 300 570
0.66 Post Inoculation & Pre-transfer/Harvest Stage N-2
Pre-Inoculation, Post- 1.) 400.sup.1 1.) 740 1.) 0.86 drain
Pre-refill 2.) 50-100.sup.1 2.) 143-228 2.) 0.17-0.26 3.) 192.sup.2
3.) 385 3.) 0.45 Stage N-2 Post Inoculation & 1.) 450 1.) 826
1.) 0.96 Pre-transfer/Harvest 2.) 450-900.sup.3 2.) 826-1594 2.)
0.96-1.84 3.) 960.sup.4 3.) 1696 3.) 1.96 .sup.1Pre-inoculation
volume and rolling seed inoculation volume for the 1 in 9
sub-cultivation process. .sup.2Rolling seed inoculation volume for
the 1 in 5 sub-cultivation processes. .sup.3Rolling seed post
inoculation & pre-transfer volume for the 1 in 9
sub-cultivation processes. .sup.4Rolling seed post inoculation
& pre-transfer volume for the 1 in 5 sub-cultivation
processes.
Tank Diameter
[0251] The tank diameter is altered to obtain the optimal aspect
ratio H.sub.L/T. Changes to tank internal diameter are limited by
acceptable aspect ratio and plant footprint. The tank ID is 0.864
m.
Tank Height
[0252] The tank height is determined from the maximum operating
volume, aspect ratio H.sub.L/T, freeboard straight side length,
base and top plate design. The final tank height is a compromise
value determined from volumetric contingency for foam, plant height
and acceptable impeller shaft length. The head to base tangent line
height is 2.347 m.
Freeboard Height
[0253] The freeboard height of 500 mm (293 litres or 31% v/v of the
maximum operating volume) is used for this seed bioreactor.
Head and Base Plate
[0254] The base and head plate design is ASME F&D for this seed
bioreactor.
Bioreactor Agitation Requirement
[0255] Agitation of the bioreactor is to achieve rapid mixing,
maintain homogeneity, maintain mammalian cells in suspension and
gas bubble dispersion. The underlying issue with achieving the
above objectives is to minimise cell damage through shear forces
originating from impeller geometry and "eddies" or vortices created
behind the impeller blades. A compromise of the above objectives
was achieved by selection of an appropriate impeller type and
gassing strategy.
Bottom Versus Top Driven Shaft
[0256] The motor drive is top mounted for the benefits as already
highlighted.
Baffles
[0257] The baffle requirement for a centre mounted impeller is
critical to prevent vortex formation. The critical issues related
to baffles are baffle number, baffle width (W), baffle length
(H.sub.baffle) and baffle to tank wall clearance (W.sub.c).
[0258] Four equally spaced baffles that are 0.1.times.T or 86 mm
wide 1.1.times.H-H.sub.h or 2099 mm tall and have a baffle to tank
wall clearance, W.sub.c of 0.01.times.T or 9 mm were used.
[0259] The thickness of baffle is not specified but the thickness
needs to ensure rigidity to the radial component of the fluid flow.
Additionally thickness needs to ensure the baffle plates are not
warped during SIP thereby affecting the baffle to tank wall
clearance.
Impeller Type, Size and Number
[0260] The impellers for the 1000 l bioreactor should be identical
formed to the 20 000 litre vessel with an identical D/T ratio.
Therefore the bottom impeller is a Lightnin's A315 at 381 mm
diameter and the top impeller is a Lightnin's A310 at 381 mm
diameter.
The Impeller Spacing, D.sub.c, D.sub.s and D.sub.o
[0261] The impeller spacing (D.sub.s) between the centre-line of
the top impeller and the centre-line of the bottom impeller is
2.times.D.sub.bottom (762 mm). The off bottom impeller clearance
(D.sub.c) is 0.4.times.D.sub.bottom (152 mm). This allows the
bottom impeller to remain submerged with liquid cover (D.sub.o) of
0.5.times.D.sub.bottom or 190 mm at the lowest post-inoculation
volume of 167 litres and both impeller submerged at 616 litres with
liquid head above the upper impeller, D.sub.o, of
0.5.times.D.sub.top (190 mm).
[0262] Table 19 highlights volumes that will form liquid surfaces
or lower liquid cover above the impeller Agitation can be modified
to avoid foaming at these critical volumes.
TABLE-US-00019 TABLE 19 Key operating volumes that cause
interaction with impellers and liquid surface Interaction Volume, L
Potential Operation Submerge top impeller with 616 Volume seen
during 0.5D.sub.A310 liquid cover inoculation of 1 in 5 processes
and rolling operation of the 1 in 9 process Liquid surface touching
top 512 Volume seen during edge of top impeller inoculation of 1 in
5 processes and rolling operation of the 1 in 9 process Liquid
surface touching bottom 492 Volume seen during edge of top impeller
inoculation of 1 in 5 processes and rolling operation of the 1 in 9
process Submerge bottom impeller 167 Volume seen during with
0.5D.sub.A315 liquid cover inoculation of 1 in 5 processes and
rolling operation of the 1 in 9 process Liquid surface touching top
90 Volume seen during rolling edge of lower impeller operation of
the 1 in 9 process Liquid surface touching bottom 21 Volumes seen
during edge of lower impeller pre-inoculation fill of 1 in 5 and 1
in 9 processes.
[0263] The 1000 l bioreactor operates at two discrete
post-inoculation volumes with either the bottom impeller submerged
during the 1 in 5 processes and 1 in 9 processes or with both
impellers submerged during the N-2 phase of the 1 in 5 process and
rolling seed operations for both 1 in 5 and 1 in 9 processes.
[0264] Table 20 shows the liquid cover above the upper and lower
impeller during operation of the 1 in 5 and 1 in 9 sub-cultivation
processes. During rolling operation of the 1 in 5 and 1 in 9
processes the liquid cover above the lower impeller falls below
0.5.times.D. It is therefore important to reduce the agitation
rate, to avoid surface gas entrainment, whilst operating at this
low volume. At 960 litres a liquid cover, (D.sub.o) of
2.05.times.Dtop is obtained. At this level K.sub.La has been shown
not to be adversely affected and bulk blending is not an issue.
TABLE-US-00020 TABLE 20 Key operating volumes and the liquid cover
above top impeller, Do and bottom impeller, D.sub.Bo Cylinderical
Do as D.sub.Bo as Operating height, Do, D.sub.Bo, ratio ratio
volume, L H (mm) mm mm of D.sub.A310 of D.sub.A315 Pre-Inoculation,
334 -- 332 -- 0.87D.sub.A315 250 L Pre-Inoculation, 590 -- 588 --
1.54D.sub.A315 400 L Post-Inoculation and 419 -- 417 --
1.10D.sub.A315 Pre-Transfer, 300 L Post-Inoculation and 675 -- 673
-- 1.77D.sub.A315 Pre-Transfer, 450 L Post drain, pre-bulk 235 --
233 -- 0.61D.sub.A315 192 L Post drain, pre-bulk 0-78 -- 76 --
0.2D.sub.A315 50-100 L Post-Inoculation and 1443 679 --
1.78D.sub.A310 -- Pre-Transfer, 900 L Post-Inoculation and 1545 782
-- 2.05D.sub.A310 -- Pre-Transfer, 960 L .sup.1Off bottom impeller
clearance, Dc = 152 mm (0.4D.sub.A315), Impeller separation, Ds =
762 mm (2D.sub.A315), tank ID of 864 mm and Height of ASME F&D
base plate, H.sub.h = 151 mm .sup.2Do = H - Ds - (Dc - H.sub.h) and
D.sub.Bo = H - (Dc - H.sub.h)
Agitation Rate--rpm, P/V and Tip Speed
[0265] Table 21 specifies the agitation rate for the 1000 litre
bioreactor. The bioreactor is agitated at around 20-260 W/m.sup.3,
preferably at 55-85 W/m.sup.3. The agitation strategy was developed
during the 500 litre pilot fermentations. An agitation rate of up
to 155.+-.1 rpm is used as an operational range.
TABLE-US-00021 TABLE 21 Agitation rate for the 1000 L bioreactor
Power per unit volume, Agitation rate, rpm W m.sup.-3 Tip Speed, m
s.sup.-1 .sup.10-155 0-150 3.1 .sup.20-145 0-145 2.9 .sup.1When
both impellers submerged .sup.2When bottom impeller submerged
Mechanical Seals Specification
[0266] A double mechanical seal that is condensate lubricated as
described was used.
Bioreactor Aeration Requirement
[0267] Table 22 shows the gas flows based upon scale up of constant
superficial gas velocity, for DOT and pH control during the
inoculum expansion in the 1000 litre bioreactor. Oxygen will not be
required for DOT control. However oxygen enriched air may be used
to facilitate lower gassing to prevent excess foaming. It is
recommended that the smaller range CA MFC should be used to
delivery nitrogen for early DOT control and reducing deviant, high
levels of DOT.
TABLE-US-00022 TABLE 22 Gas flow rate and MFC operating ranges for
the 1000 litre bioreactor Gas Operating range Comments Head
Space.sup.1 1. Clean air 1. 0-50 SLPM 1. Head space purging of
CO.sub.2 and O.sub.2 2. Nitrogen 2. Utility rated 2. For rapid DOT
probe zeroing 3. Helium 3. Utility rated 3. Tank integrity testing
Control Sparger 1. Clean air.sup.1 1. 2-20SLPM 1. Gas flow under
DOT control 2. Oxygen 2. 0.2-5SLPM 2. Gas flow under DOT control 3.
Carbon dioxide 3. 0.2-10SLPM 3. Gas flow under pH control 4.
Nitrogen.sup.2 4. 0.2-5SLPM 4. Early DOT control by ballast 5.
Helium 5. Utility rated 5. Tank integrity testing .sup.1The air and
nitrogen gas flow into bioreactor via a bypass for post SIP tank
pressurisation. .sup.2Nitrogen delivered via the 0 to 5SLPM CA MFC,
could be used during early DOT control.
[0268] The calculation of hole size and number is iterated until
the target Reynolds number of gas emerging from holes is <2000
and the Sauter mean bubble diameter for a bubble chain regime is
approximately 10 mm.
[0269] Table 23 shows the key sparger design specification for the
1000 litre bioreactor. The sparger length, S.sub.L of 305 mm is
determined for pipe geometry. The holes are distributed on either
end of the sparger to prevent bubble liberating directly under the
A315 hub. Alternatively a crescent geometry can be considered.
[0270] The pipe diameter is 25 mm. 30 2 mm holes are located on the
dorsal surface of the sparger with a single 2 mm hole located on
the ventral surface to aid free CIP drainage of the sparger.
[0271] The bioreactor port for sparger installation is designed to
fit pipe design of diameter of 25 mm. The position of port allows
the placement of control sparger at a distance of 88 mm
(D.sub.c-S.sub.c) below the bottom edge of the bottom impeller and
no greater than 64 mm from tank bottom (S.sub.c).
TABLE-US-00023 TABLE 23 Design specification for 1000 litre
bioreactor spargers Parameter Control Sparger Gas flow, SLPM 35
Number of sparge holes 30 Orifice diameter, d.sub.o, m 0.002 Gas
flow, m.sup.3 s.sup.-1 5.83E-04 Orifice area, m.sup.2 3.14E-06
Total orifice area, m.sup.2 9.42E-05 Density of air, Kg m.sup.-3
1.166 Viscosity, Nm s.sup.-2 1.85E-05 Sauter mean diameter, mm
(d.sub.vs = 1.17 V.sub.o.sup.0.4 d.sub.o.sup.0.8 g.sup.-0.2) 10.65
Gravitional acceleration, g, g m s.sup.-2 9.807 Density difference,
Kg m.sup.-3 1048.834 Reynold's number, >2000 jetting regime 782
Gas velocity at sparger, V.sub.o, m/s 6.19 Sparger length, S.sub.L,
m 0.305 Combined length to drill required holes, m 0.06 Number of
rows to fit required holes in length S.sub.L, m 1 Sparger to tank
bottom clearance, Sc, m 0.064 Sparger to bottom impeller clearance,
Dc-Sc, m 0.088
Position of Probes, Addition and Sample Ports
[0272] The design basis for positioning of probes, addition and
sample ports is the same as for the 20 000 l bioreactor.
TABLE-US-00024 TABLE 24 Probe, addition and sampling port
specification for the 1000 litre bioreactor Diameter, mm
.sup.1Position, mm Probe/Port Location (inches) (inches) Rational
Temperature Lower 38.1 (1.5'') 1. 286 (11'') Positioned to minimise
(main) ring 2. 30.degree. monitored volume Temperature Lower 38.1
(1.5'') 1. 286 (11'') Positioned to minimise (backup) ring 2.
170.degree. monitored volume PH (main) Lower 38.1 (1.5'') 1. 286
(11'') Positioned to minimise ring 2. 10.degree. monitored volume
PH (backup) Lower 38.1 (1.5'') 1. 286 (11'') Positioned to minimise
ring 2. 20.degree. monitored volume DOT (main) Lower 25.0 (0.98'')
1. 286 (11'') Positioned to minimise ring 2. 150.degree. monitored
volume DOT (backup) Lower 25.0 (0.98'') 1. 286 (11'') Positioned to
minimise ring 2. 160.degree. monitored volume Spare-2 (spare -
Lower 38.1 (1.5'') 1. 286 (11'') Positioned to minimise pCO.sub.2)
ring 2. 180.degree. monitored volume Spare-3 (spare - Lower 50.8
(2'') 1. 286 (11'') Positioned to minimise Biomass) ring 2.
190.degree. monitored volume Sample valve Lower 38.1 (1.5'') 1. 286
(11'') NovAseptic type (main) ring 2. 40.degree. Sample valve Lower
38.1 (1.5'') 1. 286 (11'') NovAseptic type (back up) ring 2.
40.degree. Alkali addition Lower 12.7 (0.5'') 1. 286 (11'')
Diametrically opposite ring 2. 190.degree. pH probes Feed 1 Lower
12.7 (0.5'') 1. 286 (11'') Diametrically opposite ring 2.
200.degree. pH probes Feed 2 Lower 12.7 (0.5'') 1. 286 (11'')
Diametrically opposite ring 2. 210.degree. pH probes Antifoam
addition Head 50.8 (2'') 1. N/A Liquid surface/0.25T plate 2.
170.degree. from tank centre Spare surface Head 50.8 (2'') 1. N/A
Liquid surface directed addition plate 2. 180.degree. to vessel
wall DOT control sparger N/A 50.8 (2'') 1. 64 (2.5'') orifice 2.
0.degree. Overlay gas Head 38.1 (1.5'') 1. N/A Diametrically
opposite plate 2. 135.degree. vent out Exhaust vent out Head 38.1
(1.5'') 1. N/A Diametrically opposite plate 2. 315.degree. overlay
gas in Transfer valve Base 50.8 (2.0'') 1. N/A NovAseptic type to
plate 2. Centre allow free draining Media inlet Head 76.2 (3'') 1.
N/A Directed into vessel plate 2. 310.degree. wall Inoculum
transfer Head 50.8 (2.0'') 1. N/A Directed into vessel from S200 to
plate 2. 320.degree. wall 1000 L CIP - Spray ball Head 76.2 (3'')
1. N/A CIP'ing of highest plate 2. 270.degree. point CIP - Spray
ball Head 76.2 (3'') 1. N/A plate 2. 60.degree. Pressure gauge Head
38.1 (1.5'') 1.) N/A plate 2.) 50.degree. Rupture disc Head 50.8
(2'') 1.) N/A plate 2.) 280.degree. Spare nozzle Head 101.6 (4'')
1.) N/A plate 2.) 160.degree. 1. Hand hole Head 1. 203.2 (8'') 1.)
N/A Single port permitting 2. Sight glass plate 2. 101.6 (4'') 2.)
70.degree. two functions Agitator shaft Head 152.4 (6'') 1.) N/A
Centre of head plate opening plate 2.) 75.degree. .sup.1Measured
from the tangential line of the base plate. Degree pertains to
plane of clockwise rotation. .sup.2Diameter of nozzle at the
bioreactor
[0273] In order to monitor, control and sample from a volume of 50
l, the probes and port ring needs to be 151 mm from tank bottom.
However the probe/port ring cannot be located this low as it falls
on the weld of the base plate and the straight cylindrical side of
the bioreactor. The probe and port ring has been specified at 286
mm from tank bottom. This permits a volume of 134 litres to be
monitored, controlled and sampled. The probes/port ring is located
as close to the tank bottom as permitted to minimise the
monitored/controlled volume.
Addition Ports, Surface and Sub-Surface
[0274] The 1000 litre bioreactor has been designed to accept two
subsurface feeds and alkali to be discharged into a well-mixed area
of the bioreactor. The foam is controlled by surface addition of 1
in 10 diluted C-emulsion. A single above surface spare addition
port directed to the vessel wall was also integrated for future
flexibility. The splashing of culture on to the surface of the
medium during inoculation of seed bioreactor should be avoided to
prevent build up of foam. Therefore the inoculum addition port is
above surface and directed to the vessel wall. The use of the
harvest port in the base plate is the ideal port for removal of
inoculum during transfer of inoculum. Additionally the medium
addition port is directed on to the vessel wall. In summary the
total addition ports are: [0275] Four surface additions with medium
inlet, inoculum inlet and a spare small addition directed to the
vessel wall and addition port for antifoam dropped on to the liquid
surface away from the vessel wall. [0276] Three subsurface
additions for feeds and alkali.
Sample Ports
[0277] The sample port design is similar to that specified for the
20 000 litre bioreactor. The sample port is located 286 mm from
tank bottom to minimise the volume that can be sampled.
Volume Measurement
[0278] The level sensor is able to measure up to 1000 litres. The
level sensor sensitivity is at least 0.25% of full span.
Bioreactor Temperature Control
[0279] The 250 to 800 litres of medium is brought to operating
temperature, typically 36.5.degree. C. during initial inoculation
and "seed rolling operation" by process control. This is achieved
by "gentle" heating of the jacket and avoid high temperature at
vessel wall.
Jacket
[0280] The bioreactor jacket area is specified with the following
considerations in mind: [0281] Steam sterilisation at
121-125.degree. C. [0282] Warming up of 250-800 litres of medium
from 10.degree. C. to 36.5.degree. C. in <2 hrs. [0283] All
points within the bioreactor must reach .+-.0.2.degree. C. of set
point, typically 36.5.degree. C. as measured by thermocouples.
[0284] Chilling of 400 litres of medium from 36.+-.2.degree. C. to
10.degree. C. in 2 hrs.
Bioreactor pH Control
[0285] The process pH is monitored and controlled with probes
connected via a transmitter to a DCS based process controller. The
process pH is controlled by addition of CO.sub.2 to bring the pH
down to set point and addition of alkali to bring pH up to set
point. Alkali is added through at least one subsurface port at
centre-line of the bottom impeller. The CO.sub.2 is added via the
control sparger.
[0286] The control and back up probes are in the lower port ring at
286 mm from tank bottom to minimise the volume that can be
monitored as shown in Table 24.
Bioreactor DOT Control
[0287] Dissolved oxygen is monitored and controlled with
polarographic DOT probe. The DOT set point maintained by
sparging:-- [0288] Initial N.sub.2 ballast and/or air on demand
[0289] Air ballast with air on demand [0290] Air ballast with
oxygen on demand
[0291] DOT control allows DOT set point to be maintained through
interchangeable use of oxygen or air as demand gas.
[0292] Control and back up probes are in the lower port ring at 286
mm from tank bottom minimise the volume that can be monitored, as
shown in table 24.
Feed Addition Control
[0293] The point of addition is 286 mm from tank bottom, in the
vicinity of the centre-line of the bottom impeller to assist in the
rapid dissipation of feed bolus.
Antifoam Addition Control
[0294] The addition point is at surface projecting 0.25.times.T
toward the tank centre or 216 mm from tank centre.
EXAMPLE 4
Bioreactor Train
[0295] The bioreactor design is based on the ability to perform
both 1 in 5 (20% v/v) and 1 in 9 (11% v/v) subculture ratios. The
bioreactor train consists of a 1000 litre (Stages N-3 and N-2) and
4000 litre (Stage N-1) seed bioreactors followed by a 20 000 litre
production bioreactor (Stage N). The operating volumes for each
bioreactor are defined in examples 1 to 3. The bioreactors are
based on a stirred tank design and a top driven agitator system was
used.
[0296] The design is based on the need to ensure a homogenous
environment with respect to process parameters such as pH,
dissolved oxygen tension (DOT) and temperature, maintaining a well
mixed cell suspension and blending nutrient feeds within the
bioreactor. This provides the necessary physicochemical environment
for optimal cell growth, product accumulation and product quality.
Key to the design philosophy is the need to maintain geometric
similarity. This allows a scale down model to be developed at 12
litre laboratory and 500 litre pilot scales. The design of the seed
and production bioreactors are based on the same principles
although some departures are required to allow for flexibility in
processing. The aspect ratios (H.sub.L/T) selected are typical of
those used in mammalian cell culture and are in the range 0.17 to
1.96 post-inoculation.
TABLE-US-00025 TABLE 25 Key bioreactor design parameters 1000 litre
4000 litre 20000 litre Aspect ratio (H.sub.L/T) 0.17-1.96 0.63-1.21
0.83-1.34 Impeller to tank 0.44-0.46 0.44-0.46 0.44-0.46 diameter
(D/T) Operating Volume 50-960 1914-3846 13913-21739 (L) Agitator
speed 0-155 0-88 0-80 (rpm) Control sparger 2-20 0-60 0-1000 CA
(SLPM) Ballast sparger No ballast sparger No ballast sparger 0-500
CA/N.sub.2 flow (SLPM) Cultivation .sup. 2-5.sup.1 2-5 10-15
residence time (days) Feed additions 2 surface 2 surface 4 surface
3 sub-surface 3 sub-surface 4 sub-surface .sup.1The culture
residence time in 1000 litre bioreactor may be higher depending on
the length of time the bioreactor is repeatedly sub-cultivated or
"rolled".
[0297] The design constraint is based upon a seeding ratio of 11%
v/v (1 in 9 dilution) and 20% v/v (1 in 5 dilution), with feed
application of 4% v/v to 25% v/v of the post-inoculation volume.
The post-inoculation volume in the production bioreactor is
adjusted for feed applications up to 15% such that after the
addition of all the feeds the final volume at harvest ends up at 20
000 l. However for feed applications greater then 15% v/v the
post-inoculation volume is adjusted for a 15% v/v feed but
following the application of feeds the final pre-harvest volume
will be a minimum of 20 000 and a maximum 22 000 litres. The
production bioreactor is expected to hold a total of 20 000 to 22
000 litres at the end of a batch. Table 26 shows the
pre-inoculation volume, inoculation volume and transfer or harvest
volume for each of the three inoculum expansion stages and the
production bioreactor.
[0298] The seed bioreactors (stage N-1 to N-3) are unlikely to be
fed therefore the maximum operating volume will be at inoculation.
The operating volume range for the 4000 litre seed bioreactor
(stage N-1) is 1914 to 3846 litres. In order to design a bioreactor
that can grow cells from 20% v/v seed split ratio, the 1000 litre
seed bioreactor (stages N-2 and N-3) will operate at two operating
ranges. For the 11% v/v seed split ratio the bioreactor train can
produce sufficient culture to meet forward processing cell
concentration criteria in a single expansion/sub-cultivation stage.
However the bioreactor train requires two expansion/sub-cultivation
stages to meet forward processing criteria for 20% v/v seed split
ratio process. Thus for 11% v/v seed split ratio process an
operating range of 400 to 450 litres is required and for the 20%
v/v seed split ratio process an operating volume range of 250 to
960 litres is required.
TABLE-US-00026 TABLE 26 Vessel sizes for bioreactor train 1000 4000
litre litre 20000 litre Stage N - 3 N - 2 N - 1 N 11% v/v Seed with
4 to 25% v/v production feed Pre-inoculation Volume (L) 400 -- 1914
15456-17096 Inoculation Volume (L) 450 -- 2153 17391-19231 Transfer
or Harvest Volume (L) 450 -- 2153 20000-21739 20% v/v Seed with 4
to 25% v/v production feed Pre-inoculation Volume (L) 250 768
2782-3077 13913-15385 Inoculation Volume (L) 300 960 3478-3846
17391-19231 Transfer or Harvest Volume (L) 300 960 3478-3846
20000-21739 Assumed operating volume Minimum Volume (L) 250 1914
13913 Maximum Volume (L) 960 3846 21739 Ratio of Maximum 3.84 2.01
1.56 volume/Minimum volume
[0299] It is recommended that the 1000 litre seed bioreactor is
inoculated from culture produced in an S200 Wave bioreactor.
[0300] 1000 l: This bioreactor is operated in batches of up to 5
days, with potential "shot additions" of feeds, for cultivation of
mammalian cells. However due to repeated drain and refill operation
at the end of each batch the total process residence time in this
bioreactor can exceed 30 days. The mammalian cells are kept in a
homogeneous suspension by agitation via an identical impeller
system to the 20 000 litre bioreactor. Additionally other features
will be kept geometrically similar to the 20 000 litre bioreactor,
where possible.
[0301] Sparging air or oxygen and air or nitrogen respectively will
control process DOT. Process pH is controlled by addition of alkali
for base control and of sparged CO.sub.2 for acid control.
[0302] The process operating volume of the bioreactor changes at
different phases of operation. Initially the bioreactor is
aseptically filled with a bolus of medium at 250 to 400 litres in
0.5 h. The bioreactor is operated in a pre-inoculation phase to
bring the process variables to predefined set points. 50 litre
culture from a (N-4) S-200 seed wave bioreactor is inoculated, by
pneumatic assisted flow, or pumped with a peristaltic pump in 25 to
30 minutes into the 1000 litre bioreactor at 1 in 5 or 1 in 9
dilutions. The post-inoculation operating volume is 300 and 450
litres for 1 in 5 and 1 in 9 seeded process respectively. The
addition of alkali for base control and 1 in 10 antifoam suspension
for suppression of foam contributes towards the final volume. The
inoculum culture may be fed by a "shot addition" if the culture
interval is longer then expected. As a result of mixing and gassing
the liquid volumes described above will expand due to gas hold up.
The extent of this rise is dependent on the sparger type used,
power per unit volume imparted by impellers and superficial gas
velocity of sparged gasses.
[0303] The N-3 stage ends when viable cell concentration reaches
transfer criteria. The N-2 stage for 1 in 5 process begins with a
bulk up in volume to 960 litre by draining of 192 litre excess
culture and addition of 768 litre fresh medium in 1.5 h. 696 to 769
litre of culture are transferred at the end of N-2 stage to the
4000 litre bioreactor for the 1 in 5 processes. For the 1 in 9
processes 239 litres are transferred to the 4000 litre
bioreactor.
[0304] The 1000 l bioreactor is continuously "drained and refilled
with fresh medium" or "rolled" to provide back up culture for the
4000 litre bioreactor. The duration of the rolling seed operation
is dependent on the length of the production campaign and the
permissible elapsed generations number of the seed culture.
Typically it is assumed that rolling seed operation is in excess of
30 days. The rolling operation consists of retaining approximately
192 litres of the 960 litre culture and diluting with 768 litre
fresh medium for the 1 in 5 processes. For the 1 in 9 processes the
1000 litre bioreactor is expected to be "rolled" by retaining 50 to
100 litre of the 450 to 900 litre culture and diluting with 400 to
800 litre fresh medium. Process control ranges are relaxed over
this operation. The medium added to the bioreactor during rolling
operation is warmed to 30.degree. C.
[0305] 4000 l: This bioreactor is operated in batch of no more then
5 days, with potential "shot additions" of feeds, for cultivation
of mammalian cells. The mammalian cells are kept in a homogeneous
suspension by agitation via an identical impeller system described
in example 1. Additionally this vessel is geometrically similar to
the 20 000 litre bioreactor.
[0306] Sparging air or oxygen and air or nitrogen respectively
controls process DOT. Process pH is controlled by addition of
alkali for base control and of sparged CO.sub.2 for acid
control.
[0307] The process operating volume of the bioreactor changes at
different phases of operation. Initially the bioreactor is
aseptically filled with a bolus of protein free medium at 1914 to
3077 litres in 1.5 h. The bioreactor operates in a pre-inoculation
phase to bring the process variables to predefined set points.
Culture from the 1000 litre (N-2) seed seed bioreactor is
inoculated by pneumatic flow at a flowrate to allow transfer in one
hour, at 1 in 5 or 1 in 9 dilutions. The post-inoculation operating
volume is 2153 to 3846 litres. The addition of alkali for base
control and 1 in 10 antifoam suspension for suppression of foam
contributes towards the final volume. The inoculum culture may be
fed by a "shot addition" if the culture interval is longer then
expected. As a result of mixing and gassing the liquid volume
expands due to gas hold up. The extent of this rise is dependent on
the sparger type used, power per unit volume imparted by impellers
and superficial gas velocity of sparged gasses.
[0308] 20 000 l: This bioreactor is operated in batch or fed batch
mode for 10 to 15 days for the cultivation of mammalian cells. The
mammalian cells are kept in a homogeneous suspension by agitation
via an impeller system.
[0309] The process operating volume of the bioreactor changes at
different phases of operation. Initially the bioreactor is
aseptically filled with cell culture medium at 13913 to 17096
litres in 1-2 h. The bioreactor is operated in a pre-inoculation
phase to bring the process variables to predefined set points.
Culture from the 4000 litre seed bioreactor (N-1) is inoculated by
pneumatic flow at a flow rate range of <4000 l/h into the 20 000
litre bioreactor at 1 in 5 or 1 in 9 dilutions. The
post-inoculation volume continuously increases following an
application of sub-surface feeds to maximum of 20 000 litres (two
feeds totaling 4 to 25% v/v). The addition of alkali for base
control and 1 in 10 antifoam suspension for suppression of foam
accounts for about 100 litres and 20 litres respectively. As a
result of mixing and gassing the liquid volume expands due to gas
hold up. The extent of this rise is depended on the sparger type
used (fluted or sintered), power per unit volume imparted by
impellers and superficial gas velocity of sparged gasses.
[0310] Table 27 describes the aspect ratios in the 20 000 litre
bioreactor at various operating volumes during normal processing.
The aspect ratios have been tested at 500 litre scale and provided
the superficial gas velocity and power per unit volume are kept
constant the K.sub.La remains constant.
TABLE-US-00027 TABLE 27 Key operating volumes and aspect ratios in
the 20 000 litre bioreactor Volume, L Liquid head, mm Aspect ratio,
H.sub.L/T Pre-Inoculation 13913-17096 2458-2977 0.88-1.07 Post
Inoculation 17391-19231 3025-3325 1.08-1.19 Harvest 20000-21739
3451-3734 1.23-1.34
* * * * *