US010179898B2

(12) United States Patent (10) Patent No.: US 10 ,179,898 B2

Khan (45) Date of Patent: * Jan . 15 , 2019
(54 ) BIOREACTOR FOR THE CULTIVATION OF 27/08 (2013.01); C12M 27 /20 (2013 .01 );
MAMMALIAN CELLS C12M 29/06 (2013.01); C12N 5/00 (2013.01);
C12N 2527/00 (2013.01)
(71 ) Applicant: Lonza Biologics PLC , Slough (GB ) (58 ) Field of Classification Search
( 72 ) Inventor: Mohsan Khan , Watford (GB ) CPC ........ C12M 27/02; C12M 27/08C12N
; C12N25275//0000;
(73 ) Assignee: Lonza Biologics PLC , Slough (GB ) USPC ........ 435/325
See application file for complete search history.
( * ) Notice : Subject to any disclaimer, the term of this
patent is extended or adjusted under 35 (56 ) References Cited
U . S .C . 154 (b ) by 0 days .
This patent is subject to a terminal dis U . S . PATENT DOCUMENTS
claimer. 4 ,378 ,436 A 3/ 1983 Heine et al.
5 ,075 ,234 A 12/ 1991 Tunac
(21) Appl. No.: 15/612,769 5 ,633 , 165 A 5 / 1997 Swartz
(Continued )
(22) Filed : Jun. 2, 2017 FOREIGN PATENT DOCUMENTS
(65 ) Prior Publication Data
CN 1326814 A 12 /2001
US 2017/0267962 A1 Sep . 21, 2017 CN 1560222 A 1/ 2005
Related U .S . Application Data (Continued )
(60 ) Continuation of application No . 14 / 885 , 802 , filed on OTHER PUBLICATIONS
Oct. 16 , 2015 , now Pat. No . 9 ,670 ,446 , which is a
division of application No . 13 / 148 ,503, filed as Bouaifi et al. “ Power consumption , mixing time and homogenisa
application No. PCT /EP2010 /000783 on Feb . 9 , tion energy in dual- impeller agitated gas -liquid reactors." Chemical
2010 . Engineering and Processing, vol. 40 (2001 ), pp . 87 -95.*
(Continued )
( 30 ) Foreign Application Priority Data
Feb . 9, 2009 (EP ) .......... 09001755 Primary Examiner — William H . Beisner
(74 ) Attorney, Agent, or Firm — Crowell & Moring LLP
(51) Int . Cl.
C12M 3 /02 (2006 .01) (57) ABSTRACT
C12M 1/00 (2006 .01)
C12M 1/ 06 ( 2006 .01) The present invention relates to large - scale bioreactors hav
C12N 5 /00 ( 2006 .01 ) ing at least two impellers , large -scale bioreactor systems and
C12M 3/ 00 ( 2006 .01) methods for the large scale cultivation and propagation of
(52 ) U . S . CI. mammalian cells using these bioreactors .
CPC ............ C12M 23 / 58 ( 2013 .01); C12M 21/ 08
(2013.01); C12M 27/02 ( 2013 .01); C12M 17 Claims, 2 Drawing Sheets

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 US 10 ,Page
179,2898 B2

(56 ) References Cited Whitford “ NSO Serum - Free Culture and Applications” , Bioprocess
International, pp . 36 -47, Dec . 2003 .
U . S . PATENT DOCUMENTS Gill et al., " Quantification of Power Consumption and Oxygen
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5 ,882,913 A 3/ 1999 Slavicek et al. dictive Fermentation Scale -Up ” , Biotechnology and Bioengineer
5 ,888 ,806 A 3 / 1999 Nguyen ing , vol. 100 , No. 6 , Aug. 15 , 2008 .
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5 , 972 ,695 A 10 / 1999 Murofushi et al. Alkaloids: Present Status and Prospects” , Journal of Natural Prod
6 ,395 ,516 B1 5 /2002 Nienow et al. ucts , vol. 56 , No. 2 , pp . 186 - 207 , Feb . 1993 .
9 ,783 ,771 B2 * 10 / 2017 Khan C12M 23 /58 International Search Report dated Mar. 24 , 2010 , issued in corre
2001/0055237 Al 12 / 2001 Kubera et al. sponding international application No. PCT/ EP2010 /000783 .
2005 /0239199 Al 10 / 2005 Kunas et al. Office Action dated Sep . 20 , 2012 in corresponding Chinese Patent
2007 / 0065927 Al 3 /2007 Brahmbhatt Application No. 201080007208 .6 (with English language transla
2007 /0172945 Al 7 /2007 O 'Kennedy et al. tion ).
2008/0068920 A1 3 /2008 Galliher et al . Ningning Ma, et al., “ Fabrication and Use of a Transient Contrac
2008 /0233631 A1 9 /2008 Higashiyama tional Flow Device to Quantify the Sensitivity of Mammalian and
2009/ 0035856 A12 / 2009 Galliher et al. Insect Cells to Hydrodynamic Forces” , Biotechnology and Bioen
2009/ 0208390 A18 / 2009 Gobby et al.
2016 /0040110 A112/ 2016 Khan gineering, Nov . 20 , 2002 , vol. 80 , No. 4 , pp . 428 -437 .
Ningning Ma, et al., “ Fabrication and Use of a Transient Contrac
FOREIGN PATENT DOCUMENTS tional Flow Device to Quantify the Sensitivity of Mammalian and
Insect Cells to Hydrodynamic Forces” , Biotechnology and Bioen
CN 1578830 A 2 /2005 gineering , Feb . 5 , 2003; vol. 81, No. 3, p . 379 .
CN 1854286 A 11 / 2006 Alvin W . Nienow , “ Reactor Engineering in Large Scale Animal Cell
EP 0025571 A 3 / 1981 Culture” , Cytotechnology , 2006 , vol. 50 , pp . 9 - 33 .
EP 0143560 10 / 1984 Frans W . J. M .M . Hoeks, " Scale -up of Stirring as Foam Disruption
EP 0477818 A 4 / 1992 (SAFD ) to Industrial Scale” , J. Ind . Microbiol Biotechnol, 2003,
EP 1 120 153 A2 8 /2001 vol. 30 , pp . 118 - 128 .
EP 1167511 A 1 / 2002 Alvin W . Nienow , “ Technical Paper : Stirred Bioreactor Engineering
EP 2039754 A 3 / 2009 for Production Scale , Low Viscosity Aerobic Fermentations : Part
2006 - 503687 2 / 2006 2 ” , 2012 (Eleven ( 11) pages ) .
2008 - 182899 8 / 2008 English translation of Japanese Office Action dated Jul. 30 , 2013
2008 -536686 9 / 2008
4260425
B2 4 / 2009 ( Three ( 3) pages ).
JP 2012 -517217 A 8 / 2012 Japanese language Office Action with English translation dated Jun .
WO WO 03 /057818 A 7 /2003 3 , 2014 (Eight ( 8 ) pages ).
WO WO 2004 /025125 A2 3 /2004 Chinese language Office Action dated Dec. 22 , 2014 (Six (6 ) pages ).
WO WO 2005/ 104706 A2 11/2005 Japanese language Office Action dated Sep . 29 , 2015 ( Six (6 )
WO WO 2007 /087438 A 8 /2007 pages).
WO WO 2007/ 129023 11/2007 Chinese language Office Action with English translation dated Feb .
WO WO 2008 /088371 A2 7 / 2008 25 , 2016 ( Fifteen ( 15 ) pages ).
Chinese language Office Action dated May 12 , 2016 (Six (6 ) pages ).
OTHER PUBLICATIONS Japanese Office Action with English translation dated Jun . 7 , 2016
( Six (6 ) pages ).
Bylund et al. “ Substrate gradient formation in the large-scale Japanese Office Action Reply dated Oct . 3, 2014 ( Twelve ( 12 )
pages ).
bioreactor lowers cell yield and increases by - product formation .” Japanese Office Action Reply dated Mar. 25, 2016 (Fifty -six (56 )
Bioprocess Engineering , vol. 18 ( 1998 ), pp . 171- 180 .* pages) .
Mahmoudi, S ., Velocity and Mixing Characteristics of Stirred European Office Action dated May 31 , 2016 ( Twelve (12 ) pages ).
Vessels With Two Impellers, Mechanical Engineering Department, Birch et al., “ Antibody Production ” , Advanced Drug Delivery
King 's College London ( 1993) . Reviews, vol. 58 (May 22 , 2006 ), pp . 671-685 .
Notice ofReasons for Rejection dated Jul. 30 , 2013 in correspond Ying Zhu et al., NSO Cell Damage by High Gas Velocity Sparging
ing Japanese Patent Application No. 2011 -548618 (with English in Protein - Free and Cholesterol- Free Cultures, Biotechnology and
language translation ). Bioengineering , vol. 101, No. 4 , Nov . 1 , 2008 , pp. 751 -760 .
Jia et al., A Bioreactor System Based on a Novel Oxygen Transfer
Method , Bioprocess International, pp . 66 -71, Jun . 2008 . * cited by examiner
 atent Jan . 15 , 2019 Sheet 1 of 2 US 10 ,179,898 B2

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U . S . Patent Jan. 15, 2019 Sheet 2 of 2 | US 10 ,179, 898 B2

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 US 10 ,179 ,898 B2
BIOREACTOR FOR THE CULTIVATION OF cells characterised in that at least one mammalian cell is
MAMMALIAN CELLS cultivated under suitable conditions and in a suitable culture
medium in a bioreactor, which has at least two impellers .
CROSS REFERENCE TO RELATED Furthermore , the present invention solves the technical
APPLICATIONS 5 problems underlying the present invention by the provision
of a bioreactor system for the cultivation ofmammalian cells
This application is a continuation application of U . S . characterised in that a ) a first bioreactor with a volumeof at
patent application Ser. No . 14 /885 ,802 , filed Oct. 16 , 2015, least 500 1 is connected with b ) a second bioreactor with a
which is a divisional application of U . S . patent application volume of at least 2000 1, which has a volume greater than
Ser. No. 13 / 148, 503 , filed Aug. 23 , 2011 , which is a national 10 the first bioreactor and wherein the second bioreactor with a
stage application of International Application No. PCT/ volume of at least 2000 1 is connected with c ) a third
EP2010 / 000783 , filed Feb . 9 , 2010 , which claims priority to bioreactor having at least two impellers and a volume of at
European Application No . 09001755.9 , filed Feb . 9, 2009 , least 10 000 1, which has a volume greater than the second
each of which is incorporated by reference in its entirety . bioreactor.
15 The present invention solves the technical problem under
FIELD OF THE INVENTION lying the present invention furthermore by the provision of
a method to cultivate and propagate mammalian cells,
The present invention relates to bioreactors and methods characterised in that a ) at least one mammalian cell is
for the large scale cultivation of mammalian cells using cultivated under suitable conditions and in a suitable culture
these bioreactors. 20 medium in a first bioreactor with a volume of at least 500 1,
b ) the medium containing the cells obtained by propagation
BACKGROUND OF THE INVENTION of the at least one mammalian cell is transferred into a
second bioreactor with a volume of at least 2000 1, c ) the
It is important in mammalian cell culture processes to transferred cells are cultivated in the second bioreactor with
maintain the physicochemical environment in view of dis - 25 a volume of at least 2000 1, d ) the medium containing the
solved oxygen , culture pH , temperature and shear sensitiv - cells obtained in step c ) is transferred into a third bioreactor
ity . Also the maintenance of the nutritional environment is with a volume of at least 10 000 1 and e ) the transferred cells
important. The maintenance of the cultivation conditions are cultivated in the third bioreactorwith a volume of at least
limits the possibility to perform large scale culturing of 10 000 1.
mammalian cells. Especially concentration gradients can 30 According to the invention, the cultivated cells are
inhibit the cell growth of mammalian cells in large -scale eukaryotic cells, preferably animal cells, more preferably
bioreactors . mammalian cells . The mammalian cells can be for example
One of the objects of the present invention is to provide human cell lines ,mouse myeloma (NSO )-cell lines , Chinese
bioreactors and methods, which allow the cultivation of hamster ovary (CHO )- cell lines or hybridoma - cell lines .
mammalian cells in large scale volumes . Furthermore , it is 35 Preferably the mammalian cells are CHO - cell lines .
an object of the present invention to provide bioreactors and Preferably the cultivated cells are used to produce anti
methods, which allow the cultivation of mammalian cells bodies , more preferably monoclonal antibodies, and /or
under optimal conditions , even if grown in large scale recombinant proteins ,more preferably recombinant proteins
volumes and therefore allow a process performance and for therapeutic use . Of course the cells may produce pep
product quality independent of the size of the bioreactor. 40 tides, amino acids, fatty acids or other useful biochemical
It is an object of the present invention to provide large - intermediates ormetabolites. According to the invention the
scale bioreactors which allow the cultivation ofmammalian target concentration of the proteins produced by the culti
cells in a homogenous environment with respect to process vated cells is more than 0 .5 g /1, preferably more than 2 .0 g/ 1
parameters such as pH , dissolved oxygen tension (DOT) and and most preferred more than 10 .0 g /1. The method accord
temperature , maintaining a well mixed cell suspension and 45 ing to the invention can be used as a batch or in a fed -batch
blending nutrient feeds within the bioreactor. process. Although the cell-culture -medium used in the
Furthermore it is an object of the present invention to method according to the invention is preferably protein free
provide devices and methods which allow the production of medium , the design does not exclude the use of protein
mammalian cells and products of the mammalian cells, containing streams.
especially proteins, peptides , antibiotics or amino acids, 50 According to the invention a bioreactor is a biocompatible
synthesised by the mammalian cells, in a large -scale manner. tank or vessel having additional equipment, for example
impellers , baffles , spargers and / or ports , which specifically
DETAILED DESCRIPTION OF THE allows for the cultivation and propagation of mammalian
INVENTION cells . Preferably the tank or vessel is in the form of a tube,
55 having on both ends of the tube , which build preferably the
The present invention solves the technical problems top and the bottom of the tank , plates . The plates are called
underlying the present invention by the provision of biore - head plate and base plate . In a particularly preferred embodi
actors , bioreactor systemsand methods for the cultivation of ment of the present invention the base plate is an American
eukaryotic cells , especially ofmammalian cells , according Society of Mechanical Engineers Flanged and Dished
to the claims. 60 (ASME F & D ) designed base plate . The head - plate design
The present invention solves the technical problem under preferably accommodates a manway or is preferably a
lying the present invention especially by the provision of a flanged head plate to allow access/removal of the impellers .
bioreactor for the cultivation of mammalian cells , charac - The total tank height is the tangential line from the inner
terised in that said bioreactor has at least two impellers . tank side of the base to the inner tank side of the head of the
Furthermore, the present invention solves the technical 65 tank .
problem underlying the present invention by the provision of The freeboard height is defined as the length of straight
a method for the cultivation and propagation of mammalian side above the liquid head when the bioreactor is filled to it's
 US 10 , 179 ,898 B2
operating volume. A minimum freeboard height is necessary Preferably , the hydrofoil impellers provide much greater
taking into account the extent of foam build up during liquid motion , resulting in a greater bulk -mixing, for a given
operation , gas hold up at maximum allowed agitation and amount of power input. This can also depend of the flow
aeration and errors in metering liquid . number (N . ).
The bioreactor according to the invention has a volume of 5 Preferably, non-hydrofoil impellers can provide liquid
preferably at least 500 1, more preferably of at least 1000 1, motion but at greater power inputs. This can have conse
more preferably of at least 4000 1, even more preferably of quences on the health of shear- sensitive mammalian cells .
at least 10 000 1, even more preferably of at least 20 000 1. In a preferred embodiment of the invention the hydrofoil
Most preferably the bioreactor according to the invention impeller is a down- flowing impeller or a up- flowing impel
has a volume of 1000 1, 1307 1, 4000 1, 5398 1, 20 000 1 or 10 ler.
27 934 1. In a preferred embodiment of the invention the top
Preferably, the bioreactor has a maximum volume of 100 impeller is a down- flowing impeller. In a preferred embodi
000 1, more preferably the bioreactor has a maximum ment of the invention the top impeller is a down - flowing
volume of 50 000 1, most preferably the bioreactor has a 15 axial hydrofoil impeller.
maximum volume of 30 000 1. In a preferred embodiment of the invention the pulling
The design of the bioreactors according to the present down characteristics of the top impeller are used to mix the
invention ensures a homogenous environment with respect well aerated liquid surface with the liquid bulk .
to process parameters such as pH , dissolved oxygen tension In a preferred embodiment of the invention the hydrofoil
(DOT) and temperature, maintaining a well mixed cell 20 impeller is a high efficiency hydrofoil impeller. In a pre
suspension and blending nutrient feeds within the bioreactor. ferred embodiment of the invention the hydrofoil impeller is
This provides the necessary physicochemical environment a Chemineer — model SC -3 impeller, a LIGHTNIN — model
for optimal cell growth , product accumulation and product A310 or A510 impeller, a Promix - model PHF series impel
quality . The design of the bioreactors according to the ler or a Cleaveland Eastern Mixers impeller.
present invention furthermore ensures the maintenance of 25 In a preferred embodiment of the invention the top
geometric similarity. This allows a scale down model to be impeller is a high efficiency hydrofoil impeller. In a pre
developed at 12 liter laboratory and 500 liter pilot scales. ferred embodiment of the invention the top impeller is a
The bioreactor for the cultivation of mammalian cells Chemineer — model SC - 3 impeller, a LIGHTNIN — model
according to the invention has at least two impellers. More A310 or A510 impeller, a Promix model PHF series impel
preferably , the bioreactor has two impellers, even more pre »30 ler The
or a Cleaveland Eastern Mixers impeller.
preferably a top impeller and a bottom impeller. top impeller is preferably a three -bladed hydrofoil
The bioreactor for the cultivation of mammalian cells design impeller, for example a A310 -type impeller from
according to the invention has preferably at least one top LIGHTNIN . The bottom impeller is preferably a four
pitched - bladed high - solidity impeller, for example of the
impeller and at least one bottom impeller , whereinn the top
3525 A315 -type from LIGHTNIN . The impeller to tank diameter
the top
impeller is preferably a hydrofoil impeller. ratio of the top impeller (D .o / T ) and /or of the bottom
The bioreactor for the cultivation of mammalian cells impeller (Dbottom / T ) is preferably at least 0 .35 and at most
according to the invention has preferably at least one top 0 .55 , more preferably at least 0 .40 and at most 0 .48, and
impeller and at least one bottom impeller, wherein the top most preferably at least 0 .44 , and at most 0 . 46 . A diameter
impeller is a hydrofoil impeller. 40 greater than 0 .5 results in disruption in axial flow , hence
The bioreactor for the cultivation of mammalian cells poor agitation and aeration .
according to the invention has preferably a volume of at least The top impeller power number ( N ) is preferably at least
1000 1 and at least one top impeller and at least one bottom 0 . 1 and at most 0 .9 , more preferably at least 0 .25 and atmost
impeller, wherein the top impeller is a hydrofoil impeller. 0 .35 , most preferably 0 .3 . The top impeller flow number
The bioreactor for the cultivation of mammalian cells 45 ( N ) is preferably at least 0 . 4 and at most 0 . 9 , more
according to the invention has preferably a volume of at least preferably at least 0 . 50 and at most 0 .60 , most preferably
4000 1 and at least one top impeller and at least one bottom 0 .56 . The bottom impeller power number ( N .) is preferably
impeller, wherein the top impeller is preferably a hydrofoil at least 0 .5 and at most 0 .9 ,more preferably at least 0 .70 and
impeller. atmost 0 . 80 ,most preferably 0 . 75 . Thebottom impeller flow
The bioreactor for the cultivation of mammalian cells 50 number (N ,) is preferably at least 0.50 and at most 0 .85 ,
according to the invention has preferably a volume of at least more preferably at least 0 .70 and at most 0 .80 , most pref
4000 1 and at least one top impeller and at least one bottom erably 0 .73 .
impeller, wherein the top impeller is a hydrofoil impeller. The impeller power number (N ) is a measure of an
In a preferred embodiment of the invention the top impeller efficiency to impart the kinetic energy of the
impeller is a hydrofoil impeller. The top impeller can be used 55 rotating impeller blades to the fluid . It is important in
preferably to provide strong bulk mixing . quantifying the gas dispersion . The impeller flow number
In a preferred embodiment of the invention the bottom (Na) is a measure of pumping ability of the impeller and is
impeller is a hydrofoil impeller. In a preferred embodiment important in quantifying fluid bulk movement.
of the invention the top impeller and the bottom impeller are The agitation rate of the at least two impellers is depen
a hydrofoil impeller. 60 dent on the scale . However , in a particularly preferred
In a preferred embodiment of the invention at least the top embodiment of the invention the agitation rate of the at least
impeller is a hydrofoil impeller. In a preferred embodiment two impellers is atmost 200 rounds per minute (rpm ),more
of the invention all impellers are hydrofoil impellers. preferably at most 165 rpm .
According to a preferred embodiment of the invention the The impeller spacing (D ) is the space between the at least
bottom impeller is a high -solidity pitch - blade impeller or a 65 two impellers . It is in a particularly preferred embodiment of
high -solidity hydrofoil impeller. The bottom impeller can be the invention at least 1x the diameter of the bottom impeller
used preferably for the dissipation of sparged gas. (Dbottom ) and at most 2xDbottom , more preferably it is
 US 10 , 179,898 B2
1, 229xDbottom or 2xDbottom . This will allow both impellers adverse shear environment. The appropriate balance
to remain submerged at the lowest post-inoculation volume. between ensuring homogenous environment that will pro
The liquid height above the upper impeller ( D .) is in a mote good cell growth and productivity ofmammalian cell
particularly preferred embodiment of the invention at least culture processes whilst minimising the adverse effects of
0 .3x the diameter of the top impeller (Dtop ) and at most 5 shear environment is dealt with in this invention . This is
2 .5xDtop .More preferably it is at least 0 .5xD top and atmost achieved through prescribing specific bioreactor geometries,
2 .0xD top impeller design and positioning , sparger design and posi
The bottom clearance (D ) is the clearance between the tioning and specific operating limits for agitation and aera
tank bottom and the centre -line of the bottom impeller. In a
particularly preferred embodiment of the invention it is at 10 tionTherates .
major damage to mammalian cells in stirred and
least 0 .35xD bottom ,more preferably it is either 0 .4xD bottom or sparged bioreactors comes from interfacial shear. Interfacial
0 .75xD bottom shear occurs as sparged gas bubbles coalesce and burst [ref:
The design of the impellers in the bioreactor according to Ma N , KoellingKW , Chalmers J J. Biotechnol Bioeng. 2002
the present invention provides optimal hydrodynamic char
acteristics in terms of bulk mixing, gas dispersion and low 15 Nov . 20 ; 80 ( 4 ): 428 -37 . Erratum in : Biotechnol Bioeng. 2003
shear. The mammalian cells are kept in a homogeneous Feb . 5 ; 81(3 ):379]. Thusminimising sparged gas flows and
suspension by agitation via the impeller system according to excessive build up of foam is desirable . The interfacial shear
the present invention . can be minimised through a combination of approaches first
The design of the impellers in the bioreactor according to by promoting surface aeration through good mixing of the
the present invention provides rapid mixing,maintain homo - 20 liquid surface with the liquid bulk and secondly higher
geneity, maintain mammalian cells in suspension and gas oxygen driving force by segregated oxygenation of cultures
bubble dispersion . The design of the impellers in the bio - through the preferably two spargers .
reactor according to the present invention minimises cell The prescribed positioning of the hydrofoil impeller,
damage through shear forces originating from impeller particularly the liquid height above the upper impeller, D .
geometry and eddies or vortices created behind the impeller 25 preferably being around 0 .5xDton , below the liquid surface
blades . can aid strong and continuous exchange of the liquid surface
In a particularly preferred embodiment of the present with the liquid bulk thereby mixing the well oxygenated
invention , the at least two impellers are a top driven agitator liquid surface with less oxygenated liquid bulk . The pre
system . scribed impeller spacing , preferably being D = 1xD bottom to
The supply of air , especially compressed air , or specific 30 2xD bottom can permit the down - flow of liquid generated by
gases, preferably oxygen , nitrogen and /or CO2 is realised the upper impeller to feed fluid flow into the lower impeller
preferably through at least one sparger. thereby ensuring the whole fluid bulk is well-mixed and
The bioreactor according to the invention has preferably separate mixing zones are notmade. The prescribed impeller
at least one sparger,more preferably the bioreactor has one bottom clearance , preferably being D = 0 . 35xD to 0 .75xD
sparger or two spargers . The bioreactor according to the 35 can ensure that the bulk flow is able to deflect off the curved
invention has preferably two spargers. Preferably the bio - ASME F & D base and rise upwards along the walls of the
reactor has at least one sparger with a pipe - geometry . bioreactor.
Preferably the at least one sparger is of the flute - type or is The segregation of the ‘on -demand oxygenated sparged
a sintered sparger. Preferably the at least one sparger is of the gas through the control sparger from the non -oxygenated
flute-type. In particularly preferred embodiment of the pres- 40 sparged gases (such as CO2, air and nitrogen ballasts )
ent invention a crescent pipe is explored . The curvature of through a ballast sparger can allow greater residence time
the crescent is preferably 0 .8xD bottom . In order to aid instal- and path length ofhighly oxygenated sparge gas bubbles in
lation and removal from side ports of the bioreactor the the fluid bulk before disengaging out of the fluid bulk and
crescent circumference is preferably 240° of the complete into the headspace . This can permit greater oxygen transfer
circumference of 0 . 8xD bottom ring. 45 rates to be provided for a given volumetric mass transfer
The at least one sparger provides sufficient oxygen mass coefficient, k , ,. The residence time and path length of the
transfer ( characterised by K , a ) to meet the oxygen demand sparged gas bubbles can be extended further through speci
of the culture . The at least one sparger provides a K , a up to fying down - flowing axial hydrofoil impellers that continu
20 h - 1 for cultures reaching up to 20x10 cells per ml with ingly pull the liquid surface and liquid bulk down .
an oxygen uptake rate of 5 mmol/l per hour. Two spargers 50 The bioreactor according to the invention has preferably
used as a dual sparger system allow the removal of dissolved at least one baffle , more preferably at least two baffles . The
CO2 and control of dissolved oxygen tension (DOT). Fluted bioreactor according to the invention has most preferably
spargers offer the benefits of easier cleaning in place (CIP ) four baffles .
and sterilisation in place (SIP ), aids with dCO , stripping and Baffles are vertical radially located plates . Baffles are used
reduced operational costs as it is multiple use . Sintered 55 to prevent the formation of a funnel or vortex formation .
spargers provide higher K a values . The lower intrinsic Kya In a preferred embodiment of the invention , the length of
value with the fluted sparge design can be compensated by the at least one baffle is 1 . 1x the total straight height ( H ) of
the use of oxygen enriched air. The gas flow rates are scaled the bioreactor. The width of thebaffle ( W ) is preferably 0 .1 %
up on the basis of constant superficial gas velocity . the internal diameter of the tank ( T ) . The baffle clearance
It is important in large scale cultivation of mammalian 60 ( W ) is preferably 0 .01x the internal diameter of the tank
cells to maintain a homogenous physicochemical environ - ( T ). The height of at least one baffle (Hbane) is preferably
ment in terms of dissolved oxygen , culture pH , and tem - 1. 1x the total straight height (H ) -the height of the bioreac
perature , and dissolved CO2, nutrient and metabolite con tor -head (Hn). Therefore Hbattle is preferably calculated
centration gradients. Whilst ensuring the physicochemical according to the formula Hote = 1. 1xH - H ) .
environment is homogenous through using appropriate agi- 65 The thickness ofthe at least one baffle is not specified but
tation and aeration , it is important to ensure the selected the thickness needs to ensure rigidity to the radial compo
operating agitation and aeration conditions do not produce nent of the fluid flow . Additionally thickness needs to ensure
 US 10 , 179 ,898 B2
the baffle plates are not warped during SIP thereby affecting sparger to tank bottom clearance (S .) of the 10001bioreactor
the baffle to tank wall clearance . according to the invention is preferably at least 50 mm and
The bioreactor according to the invention has preferably at most 75 mm , more preferably the sparger to tank bottom
at least two ports for alkali addition . More preferably , the clearance is 64 mm . The sparger to bottom impeller clear
bioreactor has two ports for alkali addition . Most preferably , 5 ance ( D . - S . ) of the 1000 1 bioreactor according to the
the bioreactor has two ports for alkali addition , wherein the invention is preferably at least 75 mm and at most 100 mm ,
first port is located at the central line of the bottom impeller more preferably the sparger to bottom impeller clearance is
and the second port is located at the central line of the top 88 mm .
impeller. Preferably the pH probes are located diametrically In a preferred embodiment of the invention , the bioreactor
opposite the alkali addition points into the bioreactor. 10 has a volume of 4000 1. The head volume ( V ) of a 4000 1
In a preferred embodiment of the invention , the bioreactor bioreactor is preferably at least 3401and atmost 370 1,more
has a volume of 1000 1. The head volume (VW) of a 10001 preferably the head volume is 359 1. The base volume (V )
bioreactor is preferably at least 45 1 and at most 65 1, more of the 4000 1 bioreactor is preferably at least 340 1 and at
preferably the head volume is 55 1. The base volume ( V ) of most 370 1, more preferably the base volume is 359 1. The
the 1000 1 bioreactor is preferably at least 45 1 and at most 15 tank internal diameter ( T ) of the 4000 1 bioreactor according
65 1, more preferably the base volume is 55 1. The tank to the invention is preferably at least 1600 mm and at most
internal diameter ( T ) of the 1000 1 bioreactor according to 1650 mm ,more preferably the tank internal diameter is 1626
the invention is preferably at least 850 mm and at most 900 mm . The tank cross -sectional area ( A ) of the 4000 1 biore
mm , more preferably the tank internal diameter is 864 mm . actor according to the invention is preferably at least 1. 90 m2
The tank cross - sectional area ( A ) of the 1000 1 bioreactor 20 and at most 2 . 30 m ' , more preferably the tank cross
according to the invention is preferably at least 0 . 55 m and sectional area is 2 ,076 m². The head height ( H ) and / or the
at most 0 .65 m², more preferably the tank cross -sectional base height (H ) of the bioreactor with a volume of 4000 1
area is 0 .586 m2. The head height (HH), which is the height according to the invention is preferably at least 260 mm and
of the head - plate , and/ or the base height (H , ), which is the atmost 300 mm , more preferably the head height and /or the
height of the base -plate , of the bioreactor with a volume of 25 base height is 282 mm . The total tank height of the 4000 1
1000 1 according to the invention is preferably at least 120 bioreactor according to the invention is preferably at least
mm and at most 180 mm , more preferably the head height 2300 mm and at most 3100 mm , more preferably the total
and /or the base height is 151 mm . The total tank height of tank height is 2817 mm . The top impeller diameter (Diop )
the 1000 1 bioreactor according to the invention is preferably and /or the bottom impeller diameter (Dottom ) of the 40001
at least 2000 mm and at most 2600 mm , more preferably the 30 bioreactor according to the invention is preferably at least
total tank height is 2347 mm . The top impeller diameter 680 mm and at most 740 mm , more preferably the top
(Dtop ) and/or the bottom impeller diameter (Dbottom ) of the impeller diameter and / or the bottom impeller diameter is
1000 1 bioreactor according to the invention is preferably at 710 mm . The clearance between the tank bottom and centre
least 350 mm and atmost 400 mm , more preferably the top line of the bottom impeller (D ) is for the 4000 1 bioreactor
impeller diameter and /or the bottom impeller diameter is 35 according to the invention , preferably at least 500 mm and
381 mm . The clearance between the tank bottom and centre - at most 560 mm , more preferably the clearance is 531 mm .
line of the bottom impeller (D ) is for the 10001bioreactor The distance between the at least two impellers , also known
according to the invention, preferably at least 120 mm and as impeller separation (D ) is for the 4000 1 bioreactor,
at most 180 mm , more preferably the clearance is 152 mm . according to the invention , preferably at least 840 mm and
The distance between the at least two impellers , also known 40 at most 900 mm , more preferably the impeller separation is
as impeller separation ( D ) is for the 1000 1 bioreactor, 872 mm . The impeller shaft diameter for the 4000 1 biore
according to the invention , preferably at least 730 and at actor according to the invention is preferably at least 51 mm
most 790 mm , more preferably the impeller separation is and atmost 64 mm . If the 4000 1 bioreactor according to the
762 mm . The impeller shaft diameter for the 1000 1 biore - invention has baffles, the length of the baffles is preferably
actor according to the invention is preferably at least 102 45 at least 2200 mm and atmost 2600 mm , more preferably the
mm and at most 152 mm . If the 1000 1 bioreactor according length is 2477 mm . The width of the baffles for the 4000 1
to the invention has baffles , the length of the baffles is bioreactor according to the invention is preferably at least
preferably at least 2000 mm and at most 2400 mm , more 150 mm and atmost 180 mm , more preferably the width is
preferably the length is 2250 mm . The width of the baffles 163 mm . The baffle clearance is for the 4000 1 bioreactor
for the 1000 1 bioreactor according to the invention is 50 according to the invention preferably at least 12 mm and at
preferably at least 70 mm and at most 100 mm , more most 20 mm , more preferably the baffle clearance is 16 mm .
preferably the width is 86 mm . The baffle clearance for the The baffle height (Hbaffle ) for the 4000 1 bioreactor according
1000 1 bioreactor according to the invention is preferably at to the invention is preferably at least 2100 mm and at most
least 7 mm and at most 11 mm , more preferably the baffle 2300 mm ,more preferably the baffle height is 2195 mm . The
clearance is 9 mm . The baffle height (Hbafie) for the 10001 55 4000 1 bioreactor according to the invention has preferably
bioreactor according to the invention is preferably at least at least one sparger, more preferably it has one sparger. The
2000 mm and at most 2200 mm , more preferably the baffle at least one sparger of the 4000 1 bioreactor according to the
height is 2099 mm . The 1000 1 bioreactor according to the invention has preferably an orifice - or pore -size of at least
invention has preferably at least one sparger, more prefer- 1.5 mm and at most 2 .5 mm ,more preferably the orifice - or
ably it has one sparger. The at least one sparger of the 1000 60 pore -size is 2 mm . The orifice - or pore - number for the 4000
1 bioreactor according to the invention has preferably an 1 bioreactor according to the invention is preferably at least
orifice - or pore -size of at least 1. 5 mm and at most 2 . 5 mm , 80 and at most 120 , more preferably the orifice - or pore
more preferably the orifice - or pore -size is 2 mm . The number is 100 . The sparger length (S _) is preferably at least
orifice - or pore -number is preferably at least 20 and at most 250 mm and at most 800 mm , more preferably the sparger
40 , more preferably the orifice - or pore -number is 30 . The 65 length is 568 mm . The sparger to tank bottom clearance (S .)
sparger length (S , is preferably at least 150 mm and at most of the 4000 1 bioreactor according to the invention is
550 mm , more preferably the sparger length is 305 mm . The preferably at least 315 mm and at most 360 mm , more
 US 10 , 179,898 B2
10
preferably the sparger to tank bottom clearance is 337 mm . sparger for the 20 0001bioreactor according to the invention
The sparger to bottom impeller clearance (D - S ) of the is preferably at least 85 and atmost 115 , more preferably the
1000 1 bioreactor according to the invention is preferably at orifice - or pore -number is 100 . The sparger length (S , ) for
least 180 mm and at most 205 mm , more preferably the the control and/or the ballast sparger is preferably at least
sparger to bottom impeller clearance is 194 mm . 5 500 mm and at most 2000 mm , more preferably the sparger
In a preferred embodiment of the invention , the bioreactor length is 1077 mm . The sparger to tank bottom clearance
has a volume of 20 000 1. The head volume ( V ) of a 20 000 (S ) of the 20 000 1 bioreactor according to the invention is
1 bioreactor is preferably at least 1600 1 and atmost 2000 1, preferably for the control and/or the ballast sparger at least
more preferably the head volume is 1803 1. The base volume 560 mm and at most 620 mm , more preferably the sparger
(V ) of the 20 000 1 bioreactor is preferably at least 16001 10 to tank bottom clearance is 593 mm . The sparger to bottom
and atmost 2000 1, more preferably the base volume is 1803 impeller clearance ( D - S ) of the 20 000 1 bioreactor accord
1. The tank internal diameter ( T ) of the 20 000 1 bioreactor ing to the invention is for the control and/ or the ballast
according to the invention is preferably at least 2500 mm sparger preferably at least 300 mm and at most 340 mm ,
and at most 3000 mm , more preferably the tank internal more preferably the sparger to bottom impeller clearance is
diameter is 2794 mm . The tank cross -sectional area ( A ) of 15 320 mm . The requirement to add ballast from a separate
the 20 000 1 bioreactor according to the invention is pref - sparger, the ballast sparger, prevents dilution of oxygen or
erably at least 5 . 8 m² and atmost 6 . 5 m²,more preferably the oxygen enriched DOT demand gas with the ballast gas. This
tank cross- sectional area is 6 , 131 m². The head height (Hn) ensures the best oxygen transfer rate (OTR ), as the oxygen
and/or the base height (H ) of the bioreactor with a volume concentration gradient of the bubbles emerging from the
of 20 000 1 according to the invention is preferably at least 20 sparger is greatest. Secondly , the use of a ballast sparger
460 mm and at most 500 mm , more preferably the head allows spargers to be located at different positions to avoid
height and/ or the base height is 485 mm . The total tank impacting DOT control on delivering desired ballast for
height of the 20 000 1 bioreactor according to the invention PCO , control. The ballast sparger can be independently
is preferably at least 4800 mm and at most 5100 mm , more designed from the control sparger.
preferably the total tank height is 4933 mm . The top impeller 25 With the bioreactor design according to the invention ,
diameter (Dtop ) and /or the bottom impeller diameter different subculture ratios can be performed . In a particularly
(Dbottom ) of the 20 000 1 bioreactor according to the inven - preferred embodiment the subculture ratios performed are
tion is preferably at least 1100 mm and at most 1300 mm , subculture ratios of at least 1 in 5 (20 % v / v ) and at most 1
more preferably the top impeller diameter and/ or the bottom in 9 ( 11 % v / v ), more preferred 1 in 5 (20 % v / v ) or 1 in 9
impeller diameter is 1219 mm . The clearance between the 30 (11% v / v ).
tank bottom and centre line of the bottom impeller ( D ) is for The invention also includes a method to cultivate and
the 20 000 1 bioreactor according to the invention , preferably propagate mammalian cells, characterised in that at least one
at least 890 mm and at most 945 mm , more preferably the mammalian cell is cultivated under suitable conditions and
clearance is 913 mm . The distance between the at least two in a suitable culture medium in a bioreactor according the
impellers , also known as impeller separation ( D ) is for the 35 invention .
20 000 1 bioreactor, according to the invention , preferably at Bioreactors according to the invention include all biore
least 1200 mm and at most 1700 mm , more preferably the actors having at least two impellers and showing at least one
impeller separation is 1498 mm . The impeller shaft diameter feature or a combination of different features outlined above .
for the 20 000 1 bioreactor according to the invention is In the method according to the invention , the agitation
preferably at least 51 mm and atmost 64 mm . If the 20 000 40 rate of the at least two impellers of the bioreactor is
1 bioreactor according to the invention has baffles , the length preferably at least 55 W / m and at most 85 W /m " . Prefer
of the baffles is preferably at least 4000 mm and at most ably , air is sparged into the culture medium with a speed of
4600 mm , more preferably the length is 4365 mm . The width at least 5x10 -3 m /s, more preferably of at least 10x10 - m /s.
of the baffles for the 20 000 1 bioreactor, according to the In a particularly preferred embodiment of the present
invention , is preferably at least 260 mm and at most 290 45 invention alkali is added through two addition ports to
mm , more preferably the width is 279 mm . The baffle distribute the alkali, which are , preferably spatially sepa
clearance for the 20 000 1 bioreactor, according to the rated from each other. This ensures quicker blending of
invention , is preferably at least 20 mm and at most 35 mm , alkali in the event of long re - circulation time in the tank .
more preferably the baffle clearance is 28 mm . The baffle CO , is preferably added via a control sparger .
height (Hbatte) for the 20 000 1 bioreactor according to the 50 Alkali and / or CO2 are preferably used to regulate the pH
invention is preferably at least 3600 mm and at most 4050 of the culture -medium .
mm , more preferably the baffle height is 3882 mm . The 20 It is preferred that control and back - up probes be in the
000 1 bioreactor according to the invention has preferably at lower port ring at 913 mm from tank bottom .
least one sparger, more preferably it has two spargers. If the In a preferred embodiment of the present invention , the
20 000 1 bioreactor according to the invention has two 55 method according to the invention takes place in a bioreactor
spargers one is preferably a control sparger and one is with a volume of 1000 1. The volume of the culture medium
preferably a ballast sparger. The control sparger for the 20 used in the method using a 1000 1 bioreactor is preferably
0001bioreactor according to the invention has preferably an during the pre - inoculation 50 1 to 250 1. During the post
orifice- or pore - size of at least 3 mm and atmost 5 mm ,more inoculation the volume of the culture medium is preferably
preferably the orifice - or pore -size is 4 mm . The ballast 60 at least 300 1 and at most 960 1. In the pretransfer /harvest
sparger for the 20 0001bioreactor according to the invention phase, the volume of the culture medium in the 1000 1
has preferably an orifice - or pore -size of at least 5 mm and bioreactor is preferably at least 300 1 and at most 960 1. The
atmost 7 mm , more preferably the orifice - or pore -size is 6 minimum operating volume (V min ) in a bioreactor with the
mm . The orifice /pore number of the control sparger for the volume of 1000 1 according to the invention is preferably
20 000 1 bioreactor according to the invention is preferably 65 between 80 1 and 120 1, more preferably the minimum
at least 230 and at most 270 , more preferably the orifice - or operating volume is 100 1, the maximum operating volume
pore -number is 250 . The orifice -pore -number of the ballast ( V ) is preferably at least 900 1 and at most 1100 1, the
 US 10 , 179,898 B2
lu
maximum operating volume is more preferably 1000 1. The 1600 mm , more preferably the height of the upper probe - or
minimum stirred volume is preferably at least 230 1 and at sample -ring is 1403 mm . The height of the lower probe- or
most 255 1, more preferably the minimum stirred volume is sample-ring is preferably at least 500 mm and at most 550
245 1. The liquid height at the minimum operating volume mm , more preferably the height of the lower probe- or
Hin
( ) is in a bioreactor with a volume of 1000 1 preferably 5 sample -ring is 531 mm .
at least 210 mm and at most 240 mm , more preferably the In a preferred embodiment of the present invention , the
liquid height at the minimum operating volume is 228 mm . method according to the invention takes place in a bioreactor
The liquid height at the maximum operating volume (Hz) in with a volume of 20 000 1. The volume of the culture
a bioreactor with a volume of 1000 1 is preferably at least medium used in the method using a 20 000 1 bioreactor is
1500 mm and at most 1900 mm , more preferably the liquid 10 preferably during the pre -inoculation 13 913 1 to 17 096 1.
height at the maximum operating volume is 1764 mm . The During the post-inoculation the volume of the culture
minimum aspect ratio (Hmin / T ) is preferably at least 0 . 15 and medium is preferably at least 17 391 1 and at most 19 231 1.
at most 0 . 19 , more preferably the minimum aspect ration is In the pretransfer /harvest phase , the volume of the culture
0 . 17 . The maximum aspect ratio ( H , - T ) for the bioreactor medium in the 20 0001 bioreactor is preferably at least 20
with a volume of 1000 1 used in a method according to the 15 000 1 and atmost 21 739 1. The minimum operating volume
invention is preferably at least 1 .8 and at most 2 .1 , more (V min ) in a bioreactor with the volume of 20 000 1 according
preferably the maximum aspect ratio is 1. 96 . The freeboard to the invention is preferably between 9000 1 and 16 000 1,
volume is preferably at least 270 1 and at most 310 1, more more preferably the minimum operating volume is 13 000 1,
preferably the freeboard volume is 293 1. The freeboard the maximum operating volume (V ) is preferably at least 19
height is preferably at least 450 mm and at most 550 mm , 20 000 1 and at most 25 000 1, the maximum operating volume
more preferably the freeboard height is 500 mm . The total is more preferably 22 000 1. The minimum stirred volume is
straight height (H ) is preferably at least 1900 mm and at preferably at least 81001 and at most 85001, more preferably
most 2200 mm , more preferably the total straight height is the minimum stirred volume is 8379 1. The liquid height at
2045 mm . The height of the upper probe - or sample -ring is theminimum operating volume (Hmin ) is in a bioreactor with
preferably at least 900 mm and at most 1200 mm , more 25 a volumeof 20 0001preferably at least 2100 mm and atmost
preferably the height of the upper probe - or sample-ring is 2500 mm , more preferably the liquid heightat theminimum
1093 mm . The height of the lower probe- sample ring is operating volume is 2309 mm . The liquid height at the
preferably at least 152 mm and at most 286 mm . maximum operating volume ( H , ) in a bioreactor with a
In a preferred embodiment of the present invention , the volume of 20 000 1 is preferably at least 3550 mm and at
method according to the invention takes place in a bioreactor 30 most 3950 mm , more preferably the liquid height at the
with a volume of4000 1. The volume of the culturemedium maximum operating volume is 3777 mm . The minimum
used in the method using a 4000 1 bioreactor is preferably aspect ratio (H min / T ) is preferably at least 0 .70 and at most
during the pre - inoculation 1914 1 to 3077 1. During the 0 .99, more preferably the minimum aspect ration is 0 .83 .
post-inoculation the volume of the culture medium is pref- The maximum aspect ratio ( H - T ) for the bioreactor with a
erably at least 2153 1 and at most 3846 1. In the pretransfer / 35 volume of 20 000 1 used in a method according to the
harvest phase , the volume of the culture medium in the 4000 invention is preferably at least 1 .2 and at most 1 .5 , more
1 bioreactor is preferably at least 2153 1 and at most 3846 1. preferably the maximum aspect ratio is 1 . 35 . The freeboard
The minimum operating volume (V min ) in a bioreactor with volume is preferably at least 5750 1 and at most 6500 1, more
the volumeof 4000 1 according to the invention is preferably preferably the freeboard volume is 6131 1. The freeboard
between 1500 1 and 2200 1, more preferably the minimum 40 height is preferably at least 900 mm and at most 1100 mm ,
operating volume is 1900 1, themaximum operating volume more preferably the freeboard height is 1000 mm . The total
( V ) is preferably at least 3800 1 and at most 4200 1, the straight height ( H ) is preferably at least 3700 mm and at
maximum operating volume is more preferably 4000 1. most 4100 mm , more preferably the total straight height is
The minimum stirred volume is preferably at least 15001 3968 mm . The height of the upper probe - or sample -ring is
and at most 1800 1, more preferably the minimum stirred 45 preferably at least 2200 mm and at most 2650 mm , more
volume is 1654 1. The liquid height at the minimum oper - preferably the height of the upper probe - or sample - ring is
ating volume (Hmin ) is in a bioreactor with a volumeof 4000 2411 mm . The height of the lower probe - or sample-ring is
1 preferably at least 800 mm and at most 1200 mm , more preferably at least 880 mm and at most 940 mm , more
preferably the liquid height at the minimum operating vol preferably the height of the lower probe - or sample - ring is
ume is 1024 mm . The liquid height at the maximum oper - 50 913 mm .
ating volume (H ) in a bioreactor with a volume of 40001 For a bioreactor with a volume of 20 000 1 the preferred
is preferably at least 1800 mm and atmost 2200 mm , more seeding ratio used is 11 % v /v ( 1 in 9 dilution ) or 20 % v / v (1
preferably the liquid height at the maximum operating in 5 dilution ), with a preferred feed application of 4 % v / v to
volume is 2034 mm . The minimum aspect ratio (Hmin / T ) is 25 % v /v of the post- inoculation volume. The post- inocula
preferably at least 0 .55 and atmost 0 .75 ,more preferably the 55 tion volume in the 20 000 1 bioreactor is preferably adjusted
minimum aspect ration is 0 .63. The maximum aspect ratio for feed applications up to 15 % such that after the addition
( H , - T ) for the bioreactor with a volume of 4000 1 used in of all the feeds the final volume at harvest ends up at 20 000
a method according to the invention is preferably at least 1 . 1 1. However, for feed applications greater then 15 % v / v the
and at most 1.4 , more preferably the maximum aspect ratio post- inoculation volume is preferably adjusted for a 15 % v / v
is 1.25 . The freeboard volume is preferably at least 850 1 and 60 feed but following the application of feeds the final pre
at most 1250 1, more preferably the freeboard volume is harvest volume will be a minimum of 20 000 1 and a
1039 1. The freeboard height is preferably at least 450 mm maximum 22 000 1. The 20 000 1 bioreactor is expected to
and at most 550 mm , more preferably the freeboard height hold a total of 20 000 1 to 22 000 1 at the end of a batch .
is 500 mm . The total straight height (H ) is preferably at least The bioreactor with a volume of 20 000 1 is preferably
2000 mm and at most 2400 mm , more preferably the total 65 operated in batch or fed batch mode for 10 to 15 days.
straight height is 2252 mm . The height of the upper probe The invention also includes a bioreactor system for the
or sample - ring is preferably at least 1200 mm and at most cultivation ofmammalian cells characterised in that a ) a first
 US 10 , 179,898 B2
13 14
bioreactor with a volume of at least 500 1, preferably of at In a preferred embodiment of the invention , the method is
least 1000 1, is connected with b ) a second bioreactor with characterised in that at least one of the bioreactors used is a
a volume of at least 2000 1, preferably of at least 4000 1, bioreactor according to the invention , more preferably all
which has a volume greater than the first bioreactor and bioreactors used are bioreactors according to the invention .
wherein the second bioreactor with a volume of at least 2000 5 Bioreactors according to the invention are in this context
1, preferably of at least 4000 1, is connected with c ) a third all bioreactors described in this description , in the examples
bioreactor according to the invention having a volume of at and in the claims.
least 10 000 1, preferably of at least 20 000 1, which has a The bioreactor of step e ) is preferably operated in batch or
volume greater than the second bioreactor. fed batch mode . The cells are cultivated in step e ) preferably
In a preferred embodiment of the invention , the bioreactor 10 for 10 to 15 days.
system is characterised in that at least one of the bioreactors Step a ) is also called stage N - 3 and/or N - 2 . Step c ) is also
is a bioreactor according to the invention .More preferably, called stage N - 1. Step e ) is also called stage N .
all of the bioreactors of the bioreactor system are bioreactors Preferably the cultivation conditions in the bioreactors of
according to the invention . steps a ), c ) and e ) are the same. More preferably, the
Bioreactors according to the invention are in this context 15 cultivation conditions in the bioreactors of steps a ), c ) and e )
all bioreactors described in this description, in the examples have a homogenous environment with respect to process
and in the claims.
The bioreactor system according to the invention is also parameters such as pH , dissolved oxygen tension and tem
perature . Preferably pH , dissolved oxygen tension and tem
called bioreactor train or device .
The bioreactor train comprises preferably different biore 20 perature
In a
in the bioreactors of steps a ), c ) and e ) are the same.
preferred embodiment of the invention , the seeding
actors, which are also called stage . The bioreactor with a ratio after the transfer steps b ) and/or d ) is at least 10 % v / v,
volume of at least 500 1 , preferably of at least 1000 1
corresponds to stage N - 3 and /or N - 2 . The bioreactor with a more preferably at least 11 % v /v (1 in 9 dilution ) and atmost
volume of at least 2000 1, preferably of at least 4000 1 30 % v /v , more preferably 20 % v / v ( 1 in 5 dilution ).
corresponds to stage N - 1 . The bioreactor with a volume of Preferably either the total medium or only a part of the
at least 10 000 1, preferably of at least 20 000 1 corresponds 2525 medium are transferred in steps b ) and d ).
Further preferred embodiments of the present invention
to stage N .
The design of the bioreactor train is based on the need to are the subject-matter of the sub claims.
ensure a homogenous environment with respect to process The present invention is illustrated in more detail in the
parameters such as pH , dissolved oxygen tension (DOT) and following examples and the accompanying figures .
temperature , maintaining a well mixed cell suspension and 30 theFIGbioreactor
. 1 shows a bioreactor according to the invention. 1 is
. 10 is the diameter of the tank ( T ). 20 is the
blending nutrient feeds within the bioreactor. The bioreac total
tors of the bioreactor train preferably show geometric simi heightstraight
of the
height of the bioreactor ( H ). 30 is the base
bioreactor ( H ). 40 is the head height of the
larity . This allows a scale - down model to develop , for bioreactor ( H ). 50 is the liquid height at the maximum
example at 12 1 laboratory scales or 500 1 pilot scales. The
35 operating
bioreactors of the stages N - 3 , N - 2 and N - 1 are used as 35 (Dion ) . 68
volume (H ). 60 is the top impeller diameter
is the top impeller. 70 is the bottom impeller
seed - bioreactors. Bioreactor of stage N is used as a produc diameter (Dhottom ) . 78 is the bottom impeller. 80 is the
tion -bioreactor. The design of the seed - and production
bioreactors is preferably based on the same principles. clearance between tank bottom and centre line of the bottom
However , some departures can be required to allow for impeller (D ). 90 is the impeller separation (D ). 100 is the
flexibility in processing. clearance of the top impeller below the liquid surface (D .).
In a preferred embodiment of the invention , the aspectest 40
40 108 is a sparger. 110 is the sparger to tank bottom clearance
ratio H , / T is at least 0 . 17 and at most 1 . 96 . ( S . ). 120 is the sparger to bottom impeller clearance (D
In a preferred embodiment of the invention there is a S .). 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 .
further bioreactor, especially a 501bioreactor corresponding FIG . 2 shows a bioreactor system of the present invention .
to stage N -4 .
In a preferred embodiment of the invention , the N - 4 4545 111
1 .1 isa avolume
with
bioreactor with a volume of 1000 1. 11 is a bioreactor
of 4000 1. 1 is a bioreactor according to the
bioreactor is a S - 200 seed wave bioreactor or a 1001 stirred invention with a volume of 20 000 1.
tank reactor
In a preferred embodiment of the invention , liquids , for EXAMPLE 1 : 20 000 L BIOREACTOR
example culture medium , can be transported from one
bioreactor to another bioreactor by pneumatic assisted flow 50 The 20 0001bioreactor is operated in batch and fed batch
or by peristaltic pumps.
The invention also includes a method to cultivate and mode for 10 to 15 days for the cultivation of mammalian
propagate mammalian cells, characterised in that a ) at least cells . The mammalian cells are kept in a homogeneous
one mammalian cell is cultivated under suitable conditions suspension by agitation via an impeller system .
and in a suitable culture medium in a first bioreactor with a se Vessel
The
Geometry
vessel geometry for the 20 000 liter bioreactor was
volume of at least 500 1, preferably with a volume of at least
1000 1, b ) the medium containing the cells obtained by determined by an iterative design basis in which the maxi
propagation from the at least one mammalian cell is trans mum working volume, freeboard straight side distance ,
ferred into a second bioreactor with a volume of at least altered aspect ratio H / T and impeller to tank diameter, D / T ratio are
2000 1, preferably with a volume of at leastbioreactor
4000 1, c with until an acceptable aspect ratio is achieved .
) the 60 Bioreactor
transferred cells are cultivated in the second bioreactor with 60 B Aspect Ratio H _/ T
a volume of at least 2000 1, preferably with a volume of at This critical design parameter allows characterisation of
least 4000 1, d ) the medium containing the cells obtained in bioreactor geometry . Tanks with higher aspect ratio offer
step c ) is transferred into a third bioreactor with a volume of longer gas residence time allowing greater Kya . However
at least 10 000 1, preferably with a volume of at least 20 000 increased head pressure can cause build up of soluble gases .
1, and e ) the transferred cells are cultivated in the third 65 Smaller aspect ratio H / T in tanks can lead to shorter gas
bioreactor with a volumeof at least 10 0001, preferably with residence time requiring greater gas flow for aeration result
a volume of at least 20 000 1. ing in greater foam build up . Impeller driven agitation to
 US 10 , 179 ,898 B2
15 16
increase Kya is also limited by H , T as surface breakage and Head and Base Plate
vortex creation will occur at lower impeller revolutions in a The selection of head and base plate design was made
low aspect ratio . Thus choice of aspect ratio is largely with a consideration for desired mechanical strength , free
experience based with some thought on issues highlighted in draining clean design and fluid flow .Maintaining consistent
table 1 . 5 plate design between scale down and full scale will contrib
TABLE 1 ute towards maintaining geometric similarity . The base plate
Summary of effect of varying aspect ratio
is of American Society of Mechanical Engineers Flanged
and Dished (ASME F & D ) design . The head - plate design
Process factor High aspect ratio Low aspect ratio accommodates a manway or a flanged head plate to allow
10 access / removal of the impellers .
Radial mixing More
Higher
effective Less effective Bioreactor Agitation Requirement
Mixing time Lower
The agitation of the bioreactor is to achieve rapid mixing ,
Oxygen transfer rate Determined by dissolved Determined by dissolved
oxygen control oxygen control maintain homogeneity , maintain mammalian cells in sus
Gas flow rate Lower Higher pension and gas bubble dispersion . The underlying issue
Cell damage Less More 15 with achieving the above objectives is minimising cell
Carbon dioxide Less effective More effective damage through shear forces originating from impeller
stripping
Pressure variations Higher Lower geometry and eddies or vortices created behind the impeller
Ease of scale up / More difficult away from More difficult away from blades . A compromise of the above objectives can be
scale down (access currently used aspect currently used aspect achieved by selection of an appropriate impeller type .
to scale data ) ratios ratios
20 Bottom Versus Top Driven Impeller Shaft
Cleanability Not affected directly by Not affected directly by
aspect ratio aspect ratio The decision to drive the agitator shaft from the top or the
Volume flexibility Less More bottom of the bioreactor is important and is determined
following a review of a number of issues highlighted in table
Table 2 describes the aspect ratios in the 20 000 liter 5 TABLE 3
bioreactor at various operating volumes during normal pro
cessing. The aspect ratios have been tested at 500 liter scale Key design issues for selection of top versus bottom entry of
and provided the superficial gas velocity and power per unit impeller shaft
volume are kept constant the K , a remains constant .
TABLE 2 Top entry Bottom entry
30
Shaft Length Long Short
Key operating volumes and aspect ratios in the 20000 litre Shaft Weight High Low
bioreactor Shaft Diameter Larger Smaller
Impeller shaft on -site Greater plant Less plant height
Volume, L Liquid head , mm Aspect ratio , H /T installation and height
removal for servicing
Pre -Inoculation 13913 - 17096 2458- 2977 0 .88 - 1 .07 35 and repair
Post Inoculation 17391- 19231 3025 - 3325 1 .08 - 1 . 19 Exposure of cell culture to No exposure Exposure
Harvest 20000 -21739 3451-3734 1.23- 1 .34 moving and stationary seal
faces
Tank Diameter 1Pressurization between Lower Higher due to the liquid
seal and vessel head
The tank diameter is altered to obtain the optimal aspect 40 Seal Lubricant leakage rate Lower Higher
ratio H _/ T . Changes to tank internal diameter ( ID ) are Base plate Design Simple Complex
limited by acceptable aspect ratio and plant footprint. The ID
positioning
Sparger to tank bottom Unrestricted Restricted
is 2 ,794 m . CIP validation Simple Complicated by sub
Tank Height merged mechanical seal
Tank height is determined from the maximum operating 45 Scale up and scale down labConsistent with Inconsistent with lab and
and pilot scale pilot scale
volume, aspect ratio H , / T , freeboard straight side length , consistency
base and top plate design . The final tank height is a com 'Pressure differential between seal and bioreactor critical for lubrication and cooling.
promise value determined from volumetric contingency for
foam , plant height and impeller shaft length . The tank height T op -entry impeller shafts tend to be longer than bottom
from base to head tan line is 4 ,933 m . 50 entry , which results in the shaft being heavier and larger
Freeboard Height diameter. Additionally the shaft length together with the
The freeboard height is defined as the length of straight inherent clearance between the two faces of the mechanical
side above the liquid head when the bioreactor is filled to it 's seal may dictate the requirement for steady bearings or
maximum operating volume. This is determined by taking stabilising ring to prevent excessive “ shaft wobble ” . Service
into account the extent of: 55 and maintenance are affected by the available space around
Foam build up during operation . the agitator, gearbox and seal assembly, and on -site shaft
Gas hold up at maximum allowed agitation and aeration . installation and removal is limited by plant height.
Errors in metering liquid . The protrusion of the seal and impeller shaft at tank
In absence of knowing the exact contribution of each with bottom restricts the placement of the sparger near the tank
piloting the process at full scale an estimate is usually made . 60 bottom . This dimension affects the tank hydrodynamics and
The amount of freeboard height is balanced with the desire therefore its amenability to change is important in specifying
to reduce the impeller shaft length for a top -driven system , an optimal design .
where extra length can complicate the design and selection The downwards load of down pumping impellers together
of available mechanical seals , the requirement for steady with the liquid head have an accumulative greater load
bearing or stabilising impeller rings. A minimum freeboard 65 (compared to up pumping or top - entry shaft) between the
height of 1000 mm ( or 6100 liter volumetric capacity or 28 % moving and stationary faces of the seal resulting in greater
V /v of the maximum operating volume) is therefore used . wear of the seal faces. Furthermore loss of over pressure in
 US 10 , 179 ,898 B2
18
the condensate line supplying the seal can result in the Impeller to Tank Diameter, D / T Ratio
culture seeping into the seal. This makes the subsurface seal
a less sanitary design .
The diameter for axial flow impellers is recommended to
The submerged seal complicates the design of a free be less than 0 .5xT. A diameter greater than this results in
draining bioreactor by compromising the position of the 5 disruption in axial flow , hence poor agitation and aeration .
harvest drain valve. Secondly the diameter of the harvest Power dissipation into the bioreactor and Reynold 's num
nozzle may be restricted thus restricting the flow rate of ber also need to be sufficiently high to maintain a turbulent
harvest stream . Therefore a top entry impeller shaft is used
(loaded ) regime. Therefore the selection of impeller diam
in the 20 000 liter bioreactor. eter is a compromise between choosing large enough diam
Baffles 10 eter to ensure adequate homogeneous mixing without
The baffle requirement for centre mounted impeller is exceeding the hydrodynamic characteristics of the bioreac
critical to prevent vortex formation . The critical issues tor. These include throttling axial flow , insufficient power
related to baffles are baffle number, baffle width ( W ), baffle dissipation , exceeding upper limits of impeller tip speed and
length (Hbaffle ) and baffle to tank wall clearance (W .). creation of poorly mixed laminar zone .
The recommendation for four equally spaced baffles that 15 Once a diameter is selected , than maintaining constant
are 0 . 1xT or 279 mm wide 1 . 1xH - Hh, or 3882 mm tall and D / T ratio is critical between scale down pilotvessels in order
have a baffle to tank wall clearance , Weof 0 .01xT or 28 mm . to maintain the central assumption of scale studies — that of
The thickness of baffle is not specified but the thickness maintaining geometric similarity .
needs to ensure rigidity to the radial component of the fluid
flow . Additionally thickness needs to ensure the baffle plates 20 The Kya scale up correlation at 12 .2 liter has been
are notwarped during SIP thereby affecting the baffle to tank determined for the four impellers at the D / T ratios shown in
wall clearance. table 4 . From a geometric similarity standpoint A310 diam
Impeller Type eter of 1 ,229 m (D / T of 0 .44 ) and A315 diameter of 1 ,285
High shear, such as Rushton (or Rushton -type), impellers m ( D / T of 0 .46 ) is recommended . However a manway
offer high power dissipation for gas dispersion but lack in 25 diameter can restrict the largest impeller diameter that can be
axial flow necessary for mixing and homogeneity . Addition installed and removed to 1,219 m . Therefore A310 and A315
ally, agitation from high shear impellers suffers from dan of to be 1 ,219 m diameter are used thereby keeping with ease
gers of excessive cell damage . impeller installation and removal and maintaining close to
Table 4 shows the impellers tested at lab scale ( 12 . 2 liter ) the geometric similarity proposed in scale down study.
that gave equivalent hydrodynamic and cell growth perfor - 30 The Impeller Clearance , D . and Spacing, D
mance . The hydrofoil is mounted above the high solidity The spacing between impellers in a bioreactor with mul
pitched blade impeller. tiple impellers is an important dimension to consider. For a
The Lightnin A310 and A315 at the D / T ratio described in bioreactor with dual Rushton turbine ( radial flow ) the
table 4 are used in the bioreactor. ungassed power consumption is equivalent to a single impel
35 ler when the dual impeller are spaced less then 0 .5xD along
TABLE 4 the shaft. At a spacing of 2xD the power consumption
Impeller types short - listed for scale down study becomes adductive . Thus efficiency of the impeller is
reduced when the impeller spacing becomes less then 0 .5xD
Impellers D /T ratio 'N , 2N , Vendor Description and the requirement formultiple impellers becomes unnec
A310 0 .44 0.30/0 .56 Lightnin Three bladed
40 essary . It is important to note that impeller spacing also
hydrofoil design impacts on the potential of creating dead zones (poorly
A315 0 .46 0.75/0 .73 Lightnin Four pitched mixed zones ) within the bioreactor. An additional constraint
bladed high on the choice of impeller spacing is discrete working vol
solidity impeller umes required within the bioreactor.
SC -3 0 .40 0 .90 /0 . 90 Chemineer Three bladed 45
hydrofoil design * The impeller spacing, Ds, of 1, 229xD bottom (1498 mm )
3HS39 0 .46 0.53/0 .58 Philadelphia Four pitched allows both impellers to remain submerged at the lowest
Mixers bladed high post- inoculation volume of 17392 liters with liquid head
solidity impeller
above the upper impeller, D , of 0 .5xDion (615 mm ) and off
'impart
N , is characteristic impeller power number. It is a measure of an impeller efficiency to
the kinetic energy of the rotating impeller blades to the fluid . It is important in 30
bottom clearance, Dc, of 0.75xDbottom (913 mm ).
quantifying the gas dispersion
N , is characteristic impeller flow number. It is a measure of pumping ability of the Table 5 highlights volumes that will form liquid surfaces
impeller and is important in quantifying fluid bulk movement. or lower liquid cover, above the impellers. Agitation needs
to be modified to avoid foaming at these critical volumes .
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 volume 17391 L
0 .5D A310 liquid cover
Liquid surface touching top 13973 Pre-inoculation volume of 13913 L liquid surface
13973
edge of top impeller breakage
Liquid surface touching 13283 Bolus addition of pre-inoculation medium will pass
bottom edge of top impeller through this liquid head
Submerge bottom impeller 8381 Bolus addition of pre-inoculation medium will pass
with 0 .5D4315 liquid cover through this liquid head
 US 10 , 179, 898 B2
19
TABLE 5 -continued
Key operating volumes that cause interaction with impellers and liquid
surface
Interaction Volume, L Potential Operation
Liquid surface touching top 5592 Bolus addition of pre-inoculation medium will pass
edge of bottom impeller through this liquid head
Liquid surface touching 3291 Bolus addition of pre-inoculation medium will pass
bottom edge of bottom through this liquid head
impeller
( ) Minimum operating volumewith lower impeller submerged is 8379 litres and minimum operating volumewith
both impellers submerged is 17399 litres
2 ) The operating volume range is 13913 to 21739 litres.

Clearance of Top Impeller Below Liquid Surface, Do . 15 TABLE 7

The breakage of the impeller blade above liquid surface is Agitation rate for the 20000 L bioreactor
undesirable as this will make the flow and power dissipation
of the impeller ineffective . In addition it will create unknown Agitation rate , rpm
Power per unit
volume, W · m -3 Tip Speed , m /s
K , a values due to significant surface entrapment of head
space gas into the fluid and excessive foam . D . is 0 .3xD for Pre Typically 28 -30 can Typically 20 can
be higher
1 .8 - 1. 9
radial flow impellers and 0 .5xD for axial flow impellers such inoculation be higher
as A310 . However as D approaches 2xD the impeller Post Typically 56 can Typically 103 , can 3 .6 can be up
inoculation be up to 80 be up to 260 to 5. 1
provides gentle blending duty . This is acceptable for the until harvest
production bioreactor application as K , a study has shown 25
that bioreactor Kya is influenced mostly by the bottom A315 Mechanical Seals Specification
impeller and the top A310 impeller contributes to bulk For bioreactor all seals are to be double mechanical seals
mixing. with a maximum “ run out” or wobble tolerance of 0 . 2 mm .
As a result of setting D and D at values D . is maintained
at an optimal range for the duration of operation of the 3030 Three types were considered ; these include:
Wet seal lubricated with sterile condensate .
production bioreactor. During the course of a batch the Dry seal lubricated with sterile gas such as N , or CA .
liquid cover above the top impeller will change from 0 .5x Non lubricated
Non lub or floating seal that are uni-rotational.
DA310 and 1.08?D4310. The liquid cover above the top All mechanical seals are recommended to be serviced on
impeller will increase as the bioreactor is fed nutrient feeds 35 an annual basis. This requires the removal of seal from the
and alkali to maintain constant pH . Table 6 shows a range of bioreactor and sending the seal assembly to the vendor.
liquid cover above the top impeller for a range of operating Therefore the design must consider ease of routine mainte
volumes . nance .
The dry type seal ( John Crane5280D type ) will produce
TABLE 6 40 3 g per year of shedding ( seal face and seal seat material)
composed of resin impregnated carbon . This is based on
Key operating volumes and the liquid cover above top impeller, Do continuous 24 hour operation over a year. The amount of
shedding for the wet seal is significantly less. Therefore a
Cylinderical wet condensate -lubricated seal is adopted for all bioreactor
45
height, H Do, mm Do as ratio double seals.
Operating volume, L mm ( inches) ( inches) of D4310 Bioreactor Aeration Requirement and Gassing Strategy
The aeration duty of the 20 000 liter bioreactor is gov
Pre -Harvest, 21739 L 3252 (128 ") 1324 (52") 1.08D4310 erned by :
Pre- Harvest , 20000 L 2968 (117" ) 1040 (41") 0.85D4310 5 0 Kya requirement.
Post- Inoculation , 19231 L 2843 (112" ) 915 (36 ") 0 .74D4310 DOT control strategy .
Post- Inoculation , 17391 L 2543 (100 ") 615 (24") 0 .5D4310 pCO2 control/ stripping strategy.
Pre -Inoculation , 15385 L 2215 (87 " ) 287 ( 11" ) 0 . 23DA310 Use of sintered or fluted spargers .
Pre -Inoculation , 13913 L 1973 (78" ) 45 (2" ) 0.04DA310 The 20 000 liter bioreactor is designed to provide Kya
55 values of up to 20h - for processes with oxygen uptake rates
(1) Off bottom impeller clearance , Dc = 913 mm ( 0 .75D4315 ), Impeller separation , Ds = of 5 mmolxL - ? xh - 7. The bioreactor design needs to be
1498 mm ( 1.229D4315 ), tank D of 2794 mm and height of ASME F & D base plate, Hy =
483 mm
flexible enough to allow cultivation of processes reaching
(2) D . - H - Ds - (De - H ;) 20x100 cellsxmL - 1.
The aeration requirement can be achieved by a number of
Agitation Rate Rpm , P /V and Tip Speed 60 different approaches . However the use of a fluted sparger
with air and oxygen enrichment to make up any deficit in
Table 7 below specifies the agitation rate for the 20 000 oxygen transfer rate (OTR ) during peak oxygen demand was
liter bioreactor. The bioreactor is agitated typically at 20 - 260 used . The advantages of this approach are :
W /m°, preferably at 55 -85 100 W /m3. The agitation strategy Easier CIP and SIP validation of fluted sparge design .
is being developed during the 500 liter pilot fermentations . 65 Larger air throughput to aid dissolved CO2 stripping.
The agitation rate of 0 to 80 + 1 rpm is therefore used as an Reduced operating cost through the avoidance of pur
operational range. chase of single use sintered elements .
 US 10 , 179,898 B2
The disadvantages of the approach selected above also A separate port for the installation of the ballast sparger
need to be considered . These include : was also built. The position of this port allows the placement
Inherent lower Kya for the low power number impellers of ballast sparger at a distance of 320 mm , (D . - S . )below the
selected . bottom edge of the lower impeller and no greater then 593
Therefore the bioreactor aeration design must have the 5 mm from tank bottom ( S .). The requirement to add ballast
from a separate sparger is due to three reasons:
flexibility to be modified to meet the desired Kya. Firstly, it prevents dilution of oxygen or oxygen enriched
Table 8 describes the gassing requirements for the 20 000 DOT demand gas with the ballast gas . This ensures the
liter bioreactor. The gas flow rates were scaled up on best OTR , as the oxygen concentration gradient of the
constant superficial gas velocity . bubbles emerging from the sparger is greatest .
Two spargers are used . The main or “ DOT control” 10 Secondly, it allows ballast sparger to be located at a
sparger supplied by dual range clean air, mass flow control different position from DOT control sparger to avoid
ler (MFC ) and oxygen MFC with gas flow metered via a impacting DOT control on delivering desired ballast for
DOT control loop and a CO , MFC metering gas via the acid pCO , control.
pH control loop . The dual range MFC 's are used to achieve Thirdly, the ballast sparger can be independently designed
precise flow control at the extreme ends of the desired from the DOT control sparger.
operating ranges . The calculation of hole size and number of holes is
The second or " ballast” sparger is supplied by a CA MFC iterated until the target Reynold 's number, Re of gas emerg
to which nitrogen is also supplied . It was measured that early ing from holes is < 2000 and the Sauter mean diameter for a
DOT control requires small nitrogen ballast to assist in early b ubble is 10 - 20 mm during chain bubble regime. Table 9
DOT demand and lower the DOT to set point. The ballast 20 shows the key specifications for the control and ballast
sparger also meters ballast air to facilitate stripping out sparger for the 20 000 liter bioreactor.
excess pCO2.
The headspace purge is used to allow removal of CO2 and TABLE 9
oxygen from the headspace . This is to facilitate better pH
and pCO , control and dilution of high oxygen blend prior to 23 — Design specification for the 20 000 litre bioreactor spargers
exhausting to environment . The ability to vary headspace DOT control Ballast
flow rate allows design of gassing strategy for various Parameter sparger sparger
processes requiring different blends of oxygen enrichment 850 500
and control point pCO2. Gas flow , SLPM
30 Number of sparge holes 250 100
Orifice diameter, do, m 0 .004 0 .006
TABLE 8 Gas flow , m . s -1 1 .42E -02 8 . 33E - 03
Orifice area , m ? 1 . 26E - 05 2 .83E -05
Gas flow rate and MFC operating ranges for the 20000 litre bio Total orifice area, m ? 3 . 14E - 03 2 .83E - 03
reactor Density of air , Kg · m - 3 1 . 166 1 . 166
Viscosity , Nm . 5 -2
35 Sauter 1 .85E - 05 1 . 85E - 05
Gas Operating range Comments mean diameter, d ,c, mm 16 . 34 19 .06
Head Space
(dys = 1.17 V .0.4 d ,0.8 g-02)
Gravitational acceleration , g m · s -2 9 .807 9 .807
Density difference , Kg .m3 1048 . 834 1048 .834
1 .) Clean air 1.) 0 - 1000 1.) Head space purging of CO2 and Reynold 's number, > 2000 jetting 1139 1117
SLPM O regime
2 .) Nitrogen 2 .) Utility rated 2.) For rapid DOT probe zeroing 40 Gas velocity at sparger, V ., m / s 4 .51 2 .95
3 .) Helium 3.) Utility rated 3.) Tank integrity testing Sparger length , SL, m 1 .077 1 .077
DOT control Combined length to drill required 1. 000 0 .6
Sparger holes, m
Number of rows to fit required holes in
1.) Clean air 1.) 10 -500 1.) Gas flow under DOT control length Sz
SLPM 45 Sparger to tank bottom clearance , Sc, m 0 .593 0 .593
2.) Oxygen 2 .) 10 - 100 2.) Gas flow under DOT control Sparger to bottom impeller clearance , 0 . 320 0 . 320
SLPM Dc- Sc , m
Carbon dioxide3 3.) 2 -150 SLPM 3.) Gas flow under pH control
Ballast Sparger
A ring sparger of 0 .8xD bottom ( 80 % diameter of bottom
1.) Clean air 1 . ) 20 - 500 1.) Variable ballast for dCO2 50 A -315 impeller diameter ) is used to distribute the holes
SLPM stripping
2 .) Nitrogenº 2 .) 20 -500 2.) Early DOT control by variable under the blades and not the impeller hub . However the CIP
SLPM flow and installation of this configuration is difficult. Therefore
selection of sparger geometry that permits distribution ofthe
The air and nitrogen gas flow into headspace enters via a bypass for post SIP tank
pressurisation . desired number of holes in a manner that is consistent with
Clean air gas flow operating range achieved by a dual CA MFC at 5-50SLPM and 55 best to distribute the holes and sanitary design can be used
50 -500 SLPM respectively .
* CO2 gas flow operating range achieved by a dual CO2 MFC at 2- 30SLPM and also .
30 - 150SLPM respectively. As an option a crescent rather then straight pipe is
4Both air and nitrogen gas flow metered from a common CA MFC .
explored . The curvature of the crescent is 0 . 8xD bottom . In
The bioreactor ports for sparger installation are designed order to aid installation and removal from side ports of the
to fit pipe design of diameter of 51 mm . The position of port 60 bioreactor the crescent circumference is 240° of the com
should allow the placement of control sparger (D . - S .) at a plete circumference of 0 .8xDbottom ring, this is 1077 mm .
distance of 320 mm below the bottom edge of the lower The DOT control sparger is 1077 mm long and has a 51
impeller and no greater 593 mm from tank bottom (S .). mm diameter. The holes have a 4 mm diameter. A total of
This results in a S value of 593 mm or (0 .65xD .) and this 250 holes divided into 2 rows (2x125 ) at 45° from the dorsal
falls outside the acceptable range of 0 .2xDe to 0 .6xDc. 65 (vertical) are used . Drain holes of 4 mm diameter on both
However hydrodynamic trials in 5001 suggest S , clearance ends of the sparger are drilled on the ventral side of the
of 0 .41 to 0 .71xD has no impact on measured Kja . sparger to aid free CIP drainage of the sparger .
 US 10 , 179 ,898 B2
23 24
The ballast sparger is 1077 mm long and of 51 mm erations included working volume range and ergonomic
diameter and has a total of 100 6 mm diameter holes in a operations . The location of probe ports, sample valve and
single dorsal row . Drain holes of 4 mm diameter on both addition points were considered together to avoid transitory
ends of the sparger are drilled on the ventral side of the spikes. Furthermore the position of the sample valve with
sparger to aid free CIP drainage of the sparger.
Position of Probes , Addition and Sample Ports respect to controlling probes needs to permit accurate esti
The probe ring position must be placed in a well-mixed mation of off- line verification of the measured process
representative region of the bioreactor. Additional consid parameter . This is shown in table 10 .
TABLE 10
Probe, addition and sampling port specification for the 20000
litre bioreactor
2 Diameter ,
mm Position, mm
Probe /Port Location ( inches) (inches ) Rational
Temperature (main ) Lower ring 38 . 1 ( 1 . 5 " ) 1 .) 9 In the plane of
2.) 30° centre- line of
bottom impeller
Temperature
(backup )
Lower ring 38 . 1 ( 1 . . 1 .) 913 ( 36 " )
2 .) 170°
In the plane of
centre -line of
bottom impeller
pH (main ) Lower ring 38. 1 (1.5 " ) 1.) 913 (36 ") In the plane of
2 .) 100 centre -line of
bottom impeller
pH (backup) Lower ring 38.1 (1.5 ") 1.) 913 (36 " ) In the plane of
2 .) 20° centre -line of
bottom impeller
DOT (main ) Lower ring 25 .0 (0 .98" ) 1.) 913 (36 " ) In the plane of
2 .) 150° centre - line of
bottom impeller
DOT (backup ) Lower ring 25 .0 (0 . 98" ) 1 .) 913 ( 36 " ) In the plane of
2.) 160° centre - line of
bottom impeller
PCO2 (spare ) Lower ring 50 .8 (2" ) tbd 1.) 913 (36 " ) In the plane of
2.) 20° centre -line of
bottom impeller
Biomass (spare ) Lower ring 50 .8 (2" ) 1.) 913 ( 36 " ) In the plane of
2 .) 160° 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° centre -line of
bottom impeller
Spare probe port Lower ring 38 . 1 ( 1 . 5 " ) 191 In the plane of
(pH -type ) 2 .) 10° centre- line of
bottom impeller
Sample valve (main ) Lower ring 12.7 (0.5" ) 1.) 913 ( 36 ") NovAseptic
2 .) 40° type
Sample valve Lower ring 12.7 (0.5") 1.) 913 (36 " ) NovAseptic
(backup ) 2 .) 50° type
Alkali addition 1 - Lower ring 50.8 (2" ) 1.) 913 (36 " ) Diametrically
Tank 1 2.) 190° opposite pH
probes
Alkali addition 2 - Centre - line 50 . 8 (2 ") 1.) 2411 (95" ) Diametrically
Tank 1 of upper 2 .) 190° opposite pH
impeller probes
Continuous feed 1 - Lower ring 50 .8 (2" ) 1 .) 913 (36 " ) Diametrically
Tank 2 2 .) 200° opposite pH
probes
Continuous feed 2 - Lower ring 50 .8 (2" ) 1 .) 913 (36 " ) Diametrically
Tank 3 2 .) 210° opposite pH
probes
DOT control sparger N / A 101.6 (4 ") 1.) 593 (23" ) Diametrically
orifice 2 .) 0° opposite ballast
sparger
Ballast sparger orifice N /A 101.6 (4 " ) 1.) 593 (23") Diametrically
2 .) 180° opposite control
sparger
Overlay gas Head plate 101.6 (4 " ) 1 .) N / A Diametrically
2 .) 1350 opposite vent
out
Exhaust vent out Impeller 50 .8 (2") 1.) N / A Diametrically
flange 2 .) 315º 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
 US 10 , 179 , 898 B2
25 26
TABLE 10 -continued
Probe, addition and sampling port specification for the 20000
litre bioreactor
2 Diameter,
mm Position, mm
Probe/Port Location (inches) (inches) Rational
Antifoam addition Head plate 50.8 (2" ) 1.) N / A Liquid surface
2 .) 170° 0 .25T
from tank
centre
Shot feed 1 - LS1 Head plate 50.8 (2") 1.) N / A
2 .) 190°
Liquid surface
Shot feed 2 - Glu Head plate 50.8 (2") 1. ) N / A Liquid surface
cose shot 2.) 180°
Small add - Spare Head plate 50.8 (2") 1. ) N / A Liquid surface -
2 .) 200° directed into
vessel wall
Media inlet Head plate 101.6 (4" ) 1 .) N / A
2 .) 310°
Nozzle directed
onto
vessel wall
Inoculum transfer Head plate 101.6 (4" ) 3.) N / A Nozzle directed
from 4000 L to 4 .) 320° onto
20000 L vessel wall
CIP - Spray ball Impeller 76 .2 (3" ) 1.) N /A CIP ’ ing of
flange 2.) 270° highest point
plate
CIP - Spray ball Head plate 76.2 (3") 5 .) N / A As per CIP
6 .) 60° design
CIP - Spray ball Head plate 76.2 (3") 1. ) N / A
2 .) 180°
As per CIP
design
CIP - Spray ball Head plate 76 .2 (3") 1. ) N / A As per CIP
2 .) 300° design
Pressure indicating Head plate 38 .1 (1.5 ") 1 .) N / A As per vessel
transmitter (PIT ) 2.) 60° vendor design
Pressure gauge Head plate 38. 1 (1.5 " ) 1 .) N / A As per vessel
2.) 50° vendor design
Rupture disc Head plate 101.6 (4 ") 1 .) N / A As per vessel
2 .) 280° vendor design
Spare nozzle Head plate 101.6 (4 " ) 1. ) N / A As per vessel
2 .) 160° vendor
design
Sight glass Head plate 101.6 (4 " ) 1.) NA As per vessel
2 .) 70° vendor
design
Light glass Head plate 76 .2 (3 ") 1.) NA As per vessel
2 .) 75° vendor
design
Manway Head plate 457.2 (18 ") 1.) N /A Personnel
2.) 90° entry
Agitator head /flange Head plate 1320 .8 (52" ) N/A Entry /removal
Impeller shaft and Agitator 304.8 ( 12 " ) N /A Entry /removal
sealmanway head /
flange
Measured from the tangential line of the base plate. Degrees pertain to plane of clockwise rotation .
Diameter of nozzle at bioreactor.

Addition Ports, Surface and Sub -Surface 50 Four subsurface additions comprised of inlets from the
The need to determine addition ports that terminate at two feed tanks and bi- level inlet from the alkali tank .
liquid surface and those that are subsurface was determined Sample Ports
by operational scenarios and the effects of feed strategy on The sample port design allows a representative sample to
process control. be taken from thebioreactor. Therefore any residualmaterial
Currently the protein free process has two continuous 55 must be as small as possible . The samples taken are used to
feeds that need to be discharged in well -mixed area of the determine off line checks for dissolved gases , pH , nutrients
bioreactor. Additional provision for glucose and an “ LS1 and biomass concentration . The orifice of the port opening
type " shot addition is also integrated in the well mixed is large enough to prevent sieving causing biomass aggre
region . The foam is controlled by surface addition of 1 in 10 gates to be retained . The 2 mm orifice NovaSeptum sam
diluted C - emulsion . The inoculation of seed into the pre - 60 pling device was used . However this has to be balanced with
inoculation bioreactor is served by avoiding build up of the desire to keep residual volume of the port low . The port
foam which will arise as the culture is dropped onto the needs to be positioned in a well -mixed zone adjacent to the
surface of the medium . Following ports were designed : probes that need to be verified by off-line checks and will be
Six surface additions with media inlet, inoculum inlet, one determined via nozzle position (see table 10 ).
small addition inlet directed into the wall of the vessel 65 Add Tanks
while the others dropped onto the liquid surface away In order to reduce cost and time the add -tanks supplying
from the tank wall. the bioreactor are of modular design . The production bio
 US 10 , 179,898 B2
27 28
reactor has three 2500 liter nominal volumes add tanks. The Bioreactor DOT Control
add tanks are filled at 25 l/min . The flow rate of feeds from Dissolved oxygen is monitored and controlled with pola
the add tanks to the bioreactor is controlled at 0 .2 to 0 1. r ographic DOT probe . The DOT set point maintained by
milliliters of feed per liter of post- inoculation bioreactor sparging:
volume per hour (ml/1/h ). It is expected that feed rate is 5 Initial N , ballast and /or air on demand .
controlled at + 5 % of set point. Air ballast with air on demand .
The production bioreactor is serviced by three 1372 mm Air ballast with oxygen on demand .
ID by 1880 mm add tanks. These tanks have the capability Reversing gas usage once oxygen demand decreases.
to be cleaned and sterilised independently and together with Cascade DOT control allows DOT set point to be main
the production bioreactor. tained through changes in the ballast and demand gas in
Manway conjunction with ramping of agitator speed .
Access into the bioreactor is required for certain service In order to control pCO , the ballast required to strip out
operations . Access can be gained by considering a flanged excess dCO , impacts DOT control. Therefore the DOT
head plate or incorporation of a manway into the head plate . 15 control is considered together with pCO , control for those
The need for access into the bioreactor is for : processes where metabolic CO2 is liberated . DOT is con
Installation of impellers. trolled at + 2 % of set point. Control and back -up probes are
Installation and replacing of impeller and impeller shaft. located in the lower port ring at 913 mm from tank bottom .
Installation and replacing of mechanical seal. Bioreactor Dissolved CO2 Control
Service of vessel furniture . 20 The process dCO2 is monitored with an pCO2 probe and
Potential modification of sparger position to obtain excess dCO , is stripped by gassing CA through the ballast
desired hydrodynamic characteristics. sparger . The optimal position for this probe is close to the pH
The size of themanway must be sufficient to allow access probes.
for the above objectives . The manway used was of sufficient Feed Addition Control
diameter to allow the removal of two impellers of 1219 mm 25 The feeds (SF22 and amino acid ) are high in pH and
diameter. osmolality . Therefore bolus additions need to be avoided to
VolumeMeasurement maintain good pH control. However the control of desired
The design ensures that any sensor gives sufficient pre flow rates ( + 5 % of set point) is technically challenging.
cision in volume measurement around the operating range. Therefore an addition strategy that encompasses point of
The volumemeasurement in bioreactor is able to measure 30 addition with delivery mode avoids the circulation of feed
a range of 13 000 to 25 000 liters . The sensor sensitivity bolus and potential variations of pH control.
needs to be at least 0 . 5 % of full span . Therefore the point of addition is in the plane of the
Volume measurement in the feed add - tanks and alkali centre - line of the bottom impeller that is 913 mm from tank
tank is able to measure 0 to 2200 and 2500 liters respec
tively . The sensor sensitivity needs to be at least 0 .2 % of full 35 Antifoamto Addition
as bottom assist in the rapid blending of feed bolus .
Control
span . This will permit hourly verification of feed flow rate at Antifoam ( C -emulsion ) addition is added as required to
the minimum flow rate of 0 . 2 mill per hour or 3 . 5 1 per hour
by measuring the volume decrease in the add tanks. maintain the bioreactor liquid surface free of foam . A
Bioreactor Temperature Control working stock of 1 in 10 diluted C - emulsion can be dosed on
The medium is brought to operating temperature and pH 40 the liquid surface . The antifoam suspension is continuously
by process control. This is achieved by “ gentle” heating of agitated in the storage container to prevent partitioning . It is
the jacket (avoid high temperature at vessel wall). The important to dose the antifoam close to the centre of the tank
temperature control range during operation is 36 to 38° C . to diminish the effects of the radial component of the fluid
with an accuracy of +0 .2° C . at set point. flow carrying the antifoam to the tank walls where it will
Jacket 45 adhere . Therefore the addition point is 0 .25xT toward the
The bioreactor jacket area is specified with the following tank centre or 699 mm from tank centre .
considerations in mind : —
Steam sterilisation at 121- 125° C . EXAMPLE 2 : 4000 LITER BIOREACTOR
Warming up of medium from 10° C . to 36 .5° C . in < 2 h .
All points within the bioreactormust reach + 0 . 2° C . of set 50 Vessel Geometry
point, typically 36 .5° C ., as measured by thermo The vessel geometry for the 4000 liter bioreactor was
couples. determined by an iterative design basis in which the maxi
Chilling of medium from 36 .5° C . to 10° C . in < 2 h . mum working volume, freeboard straight side distance
Bioreactor pH Control aspect ratio (H / T ) and impeller to tank diameter ratio (D / T )
The process pH is monitored and controlled with probes 55 are altered until an acceptable aspect ratio is achieved .
connected via a transmitter to a DCS based process control- Bioreactor Aspect Ratio H , / T
ler. The process is be controlled by addition of CO , to bring Table 11 describes the aspect ratios in the 4000 liter
the pH down to set point and addition of alkali to bring pH bioreactor at the various operating volumes during normal
up to set point. pH is controlled at + 0 .03 of set point. processing . These aspect ratios arise from the selection of
Alkali is added through two addition points to distribute 60 tank ID and the operating volume required . From a process
the alkali . This ensures quicker blending of alkali in the ing perspective the mixing requirements at the three oper
event of long recirculation time in the tank . The CO2 is ating conditions are different. During pre - inoculation stage
added via the control sparger. the bioreactor mixing is important to allow medium to
Control and back - up probes are located in the lower port equilibrate with minimal K , a requirement. However for
ring at 913 mm (see table 10 ) from tank bottom . Addition - 65 post- inoculation and pre -transfer stages both mixing and
ally the pH probes are located diametrically opposite the Ka are important considerations. Therefore both these
alkali addition points into the bioreactor. features were tested at the aspect ratio range .
 US 10 , 179 ,898 B2
29 30
TABLE 11 Baffles
Key operating volumes and aspect ratios in the 4000 litre bioreactor
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
Volume, L Liquid head , mm Aspect ratio, H [/ T 5 length (Hbafle ) and baffle to tank wall clearance (W .).
Pre - Inoculation 1.) 1914 1 .) 1031 1 .) 0 .63 Four equally spaced baffles that are 0 .1xT or 163 mm
2 .) 2782 2 .) 1448 2 .) 0 .89 wide 1. 1xH - H ,, or 2195 mm tall and have a baffle to tank
3 .) 3077 3 .) 1590 3 .) 0 . 98
wall clearance , W . of 0 .01xT or 16 mm were used .
Post Inoculation & 1.) 2153 1.) 1146 1. ) 0 . 70The thickness of the baffles is not specified but the
Pre - transfer 2 . ) 3478 2 .) 1783 2 .) 1. 10 thickness needs to ensure rigidity to the radial component of
3.) 3846 3 .) 1960 3 .) 1.21 10 the fluid flow . Additionally thickness needs to ensure the
baffle plates are not warped during SIP thereby affecting the
Tank Diameter baffle to tank wall clearance .
The tank diameter was altered to obtain the optimal aspect Impeller Type , Size and Number
ratio H , / T . Changes to tank internal diameter are limited by The impellers for this bioreactor are identically formed to
acceptable aspect ratio and plant footprint. The tank ID is 15 the 20000 liter vessel and have a identical D / T ratio of 0 .44 .
1626 mm . The bottom impeller is a Lightnin 's A315 at 710 mm of
Tank Height diameter and the top impeller is a Lightnin 's A310 at 710
Tank height is determined from the maximum operating mm of diameter.
volume, aspect ratio H , T , freeboard straight side length , The Impeller Spacing, D ., D , and D .
base and top plate design . The final tank height is a com - 20 The impeller spacing , Ds, between the centre -line of the
promise value determined from volumetric contingency for top impeller and the centre - line of the lower impeller is
foam , plant height and acceptable impeller shaft length . The 1,229xD bottom or 872 mm . The off bottom impeller clear
head to base tan line height is 2817 mm . ance , D . is 0 .75xD bottom or 531 mm . This allows the lower
Freeboard Height impeller to remain submerged at the lowest post- inoculation
The freeboard height of 500 mm (1039 liter or 27 % v /v of 25 volume of 2153 liters and both impellers submerged at 3367
the maximum operating volume) is used for this seed liters with liquid head above the upper impeller (D ) of
bioreactor. 0 .5xD on or 358 mm . top

Head and Base Plate Table 12 highlights the volumes that will form liquid
The base and head plate design is a ASME F & D design surfaces or lower liquid cover above the impeller. Agitation
for this seed bioreactor. can be modified to avoid foaming at these critical volumes .
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 of 1 in 5 processes
with 0 .5DA310 liquid
cover
Liquid surface touching 2758 Volumes seen during pre -inoculation fill of 1 in 5
top edge of top impeller processes .
Liquid surface touching 2621 Volumes seen during pre- inoculation fill of 1 in 5
bottom edge of top impeller processes.
Submerge bottom impeller Volumes seen during pre- inoculation fill of 1 in 5
1654 processes
with 0 .5D A315 liquid .
cover
Liquid surface touching 1104 Volumes seen during pre - inoculation fill of 1 in 5
top edge of bottom impeller and 1 in 9 processes .
Liquid surface touching 650 Volumes seen during pre -inoculation fill of 1 in 5
bottom edge of bottom and 1 in 9 processes .
impeller
( 1 ) Minimum operating volume with lower impeller submerged is 1654 litres and minimum operating volumewith
both impellers submerged is 3433 litres
(2 ) The operating volume range is 1914 to 3846 litres.

Bioreactor Agitation Requirement The 4000 1 bioreactor can operate at two discrete post
The agitation of the bioreactor is to achieve rapid mixing, 5555 merged
inoculation volumes with either the lower impeller sub
( during cultivation of 1 in 9 seeding process ) or with
maintain homogeneity , maintain mammalian cells in sus both impellers submerged ( during 1 in 5 seeding process ),
pension and gas bubble dispersion . The underlying issue for table 13 shows the liquid cover obtained for the upper and
achieving the above obiectives is minimising cell damage lower impeller during its operation .
A liquid
through shear forces originating from impeller geometry and 60 A315 coveris observed
impeller of 0 .67 toduring
0 .82xDbottom above
cultivation the lower
of the 1 in 9
eddies or vortices created behind the impeller blades. A seeded processes. This is within the recommendations of 0 . 5
compromise of the above objectives was achieved by selec - to 1xD .
tion of an appropriate impeller type. A liquid cover of 0 . 06 to 0.78xDiop above the top A310
Bottom Versus Top Driven Impeller Shaft impeller is observed during cultivation of 1 in 5 seeded
65 process. The lower liquid cover is outside the recommen
The motor drive is top mounted for the benefits already d ation . However this liquid cover is observed during pre
highlighted . inoculation when mixing and agitation are less critical.
 US 10 , 179 ,898 B2
31
TABLE 13
Key operating volumes and the liquid cover above top impeller, Do and bottom
impeller, DR.
Cylinderical Do , Do Do as ratio DB, as ratio
Operating volume, L height, H (mm ) mm mm of D4310 of D4315
Post - Inoculation and 863 or 34 " - 614 or 0.82D4315
Pre - Transfer, 2153 L 24 "
Post - Inoculation and 1501 or 59" 380 0 .53DA310 —
Pre - Transfer, 3478 L or
15 "
Post-Inoculation and 1678 or 66 " 557 0 .78D4310
Pre - Transfer , 3846 L or
Pre -Inoculation , 1914 L 748 or 29 " - 499 or - 0 .67DA315
20"
Pre-Inoculation, 2782 L 1166 or 452 " or - 0 .06D4310 —

Pre- Inoculation , 3077 L 1308 or 52 " 187 0.26D4310 —

or 7"
(1) Off bottom impeller clearance , Dc = 531 mm (0 .75D4315 ), Impeller separation , Ds = 872 mm
(1 .229D4315), tank D of 1626 mm and Height of ASME F & D base plate, H , = 282 mm
( ) Do = H - Ds - Dc - Hy) and DBe = H - (Dc - H )

Agitation RateRpm , P /V and Tip Speed TABLE 15 -continued

Table 14 specifies the agitation rate for the 4000 liter Gas flow rate and MFC operating ranges
bioreactor. The bioreactor will be agitated typically at 25 for the 4000 litre bioreactor
20 -260 W /m -3, preferably at 55 -85 W /m - 3. The agitation
strategy was developed during the 500 liter pilot fermenta Gas Operating range Comments
tions. An agitation rate of 0 to 88 + 1 rpm is therefore used as 3. Carbon dioxide 3. 1.0 -20SLPM 3 . Gas flow under pH control
an operational range. 4 . Nitrogen ?
304 4 . 2.0 -15SLPM 4. Early DOT control by ballast
TABLE 14 5 . Helium 5 . Utility rated 5. Tank integrity testing
Agitation rate for the 4000 L bioreactor The air and nitrogen gas flow into bioreactor via a bypass for postSP tank pressurisation .
?Nitrogen delivered via the 2 to 15SLPM N2 MFC and could be used during early DOT
control
Agitation rate , rpm Power per unit volume, W /m3 Tip Speed , m /s
10 - 88 0 - 150 0 .0 -3.3 35 The calculation of hole size and number of holes , for the
20 -86 0 - 150 0 .0 - 3. 2 fluted sparger, is iterated until the target Reynolds number of
When both impellers submerged
gas emerging from holes is < 2000 and the Sauter mean
When bottom impeller submerged bubble diameter for a bubble chain regime is approximately
10 mm .
Mechanical Seals Specification 40 Table 16 show the key sparger design specification for the
A double mechanical seal that is condensate lubricated is 4000 liter bioreactor. The sparger length , S , of 568 mm is
used as described . determined for pipe geometry . The holes are distributed on
Bioreactor Aeration Requirement either end of the sparger to prevent bubble liberating directly
Table 15 shows the gas flow , based upon scale up of under the A315 hub . Alternatively a crescent geometry can
constant superficial gas velocity , for DOT and pH control 43 be used . The pipe diameter is selected to aid spacing of the
during the inoculum expansion in the 4000 liter bioreactor. desired number of holes . The diameter is 38 mm . The 100
Oxygen is not required for DOT control. However oxygen 2 mm holes are located on the dorsal surface of the sparger
enriched air can be used to facilitate lower gassing to prevent with a single 2 mm hole located on the ventral surface to aid
excess foaming. It is recommended that a smaller range N free CIP drainage of the sparger.
MFC should supply nitrogen for early DOT control and 50 The bioreactor port for sparger installation is designed to
reducing deviant, high levels of DOT. a fit pipe design of diameter of 38 mm . The position of the
port allows the placement of a control sparger at a distance
TABLE 15 of 194 mm , D .- S . below the bottom edge of the lower
Gas flow rate and MFC operating ranges 55 impeller
impell and no greater 337 mm from tank bottom , Sc.
for the 4000 litre bioreactor
TABLE 16
Gas Operating range Comments
Design specification for the 4000 litre bioreactor sparger
Head Space !
Parameter Control Sparger
1 . Clean air 1. 0 -200SLPM and 60
1. Head space purging of CO2 and
02 Gas flow , SLPM 105
2 . Nitrogen 2 . Utility rated 2 . For rapid DOT probe zeroing Number of sparge holes 100
3 . Helium 3. Utility rated 3. Tank integrity testing Orifice diameter, do, m 0 .002
Control Sparger Gas flow , m . s - 1 1 . 75E - 03
Orifice area , m ? 3 . 14E - 06
1. Clean air? 1. 10 -60SLPM 1. Gas flow under DOT control 65 Total orifice area , m ? 3 . 14E - 04
2 . Oxygen 2 . 1 .0 - 10SLPM 2 . Gas flow under DOT control Density of air , Kg . m -3 1 . 166
 US 10 , 179 ,898 B2
34
TABLE 16 -continued TABLE 16 -continued
Design specification for the 4000 litre bioreactor sparger Design specification for the 4000 litre bioreactor sparger
Parameter Control Sparger Parameter Control Sparger
Viscosity, Nm : s -2 1 .85E - 05 Number of rows to fit required holes in length S
Sauter mean diameter , mm 10 .21 Sparger to tank bottom clearance , Sc , m 0 .337 (13 ")
(dus = 1.17 V .0.4 d .0.8 g -0.2) Sparger to bottom impeller clearance, Dc-Sc , m 0 . 194 (8 " )
Gravitional acceleration , g, m · s - 2 9 . 807
Density difference , Kg .m3 1048 .834
Reynold 's number, > 2000 jetting regime 704 10 Position of Probes, Addition and Sample Ports
Gas velocity at sparger , V ., m /s 5 .57
Sparger length , S , , m 0 .568 The design basis for positioning of probes, addition and
Combined length to drill required holes , m 0 .2 sample ports has been covered in example 1 and are listed
in table 17 :
TABLE 17
Probe, addition and sampling port specification for the 4000 litre bioreactor
2Diameter, 'Position,
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° centre -line of
bottom impeller
Temperature Lower ring 38.1 (1.5" ) 1.) 531 (21" ) In the plane of
(backup ) 2 .) 170° centre- line of
bottom impeller
pH (main ) Lower ring 38. 1 (1.5" ) 1.) 531 (21") In the plane of
2 . ) 10° centre -line of
bottom impeller
pH (backup) Lower ring 38. 1 (1.5 ") 1.) 531 (21" ) In the plane of
2 .) 20° centre -line of
bottom impeller
DOT (main ) Lower ring 25.0 (0 .98" ) 1.) 531 (21" ) In the plane of
2 .) 150° centre- line of
bottom impeller
DOT (backup ) Lower ring 25 .0 (0 .98" ) 1.) 531 (21" ) In the plane of
2.) 160° centre-line of
bottom impeller
Spare- 1 (nutrient) Lower ring 25.0 (0 . 98 ") 1.) 531 (21" ) In the plane of
2.) 1700 centre - line of
bottom impeller
Spare-2 (pCO2) Lower ring 38. 1 (1.5 " ) 1.) 531 (215 ) In the plane of
2 .) 180° centre -line of
bottom impeller
Spare- 3 (biomass ) Lower ring 50.8 (2" ) 1.) 531 (21" ) In the plane of
2 .) 190° centre -line of
bottom impeller
Sample valve Lower ring 12 . 7 (0 1.) 531 (21" ) NovAseptic type
(main ) 2 .) 40°
Alkali addition Lower ring 50.8 (2 ") 1.) 531 (21" ) Diametrically
2.) 190° opposite pH
probes
Feed 1 Lower ring 50 .8 (2" ) 1.) 531 (21" ) Diametrically
2.) 200° opposite pH
probes
Feed 2 Lower ring 50 .8 (2") 1.) 531 (21" ) Diametrically
2 .) 210° opposite pH
probes
Antifoam addition Head plate 50 .8 (2" ) 1.) N / A Liquid surface /
2.) 170° 0 .25T from tank
centre
Spare surface addition Head plate 50 .8 (2" ) 3.) N / A Liquid surface
4 .) 180° directed to
vessel wall
DOT control sparger N / A 50. 8 (2") 337 ( 13 " )
orifice 1.) 0°
Overlay gas Head plate 101.6 (4 ") 1 .) N / A Diametrically
2 .) 135° opposite vent out
Exhaust vent out Head plate 50 .8 (2" ) 1 .) N / A Diametrically
2 .) 315° 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° vessel wall
 US 10 , 179, 898 B2
35 36
TABLE 17 -continued
Probe. addition and sampling port specification for the 4000 litre bioreactor

2Diameter, Position ,
Probe/ Port Location mm (inches ) mm (inches ) Rational
Media inlet Head plate 10 3.) N / A Directed into
4 .) 310° vessel wall
CIP - Spray ball Impeller 76 1 .) N / A CIP ’ ing of
flange plate 2 .) 270° highest point
CIP - Spray ball Head plate 76.2 (3") 1. ) N / A
2 .) 60°
CIP - Spray ball Head plate 76.2 (3") 1 .) N / A
2 .) 180°
CIP - Spray ball Head plate 76 .2 (3") 1 .) N / A
2 .) 300°
Pressure indicating Head plate 38 .1 (1.5 ") 1.) N / A
transmitter (PIT ) 2 .) 60°
Pressure gauge Head plate 38 .1 (1.5" ) 1.) N / A
2 .) 500
Rupture disc Head plate 101.6 (4" ) 1. ) N / A
2 . ) 280°
Spare nozzle Head plate 101.6 (4" ) 1 .) N / A
2 .) 160°
Sight glass Head plate 101.6 (4") 1 .) N / A
2 .) 70°
Light glass Head plate 76.2 (3") 21 ..)) 750N / A
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
Measured from the tangential line of the base plate. Degrees pertain to plane of clockwise rotation .
Diameter of nozzle at bioreactor.

Addition Ports, Surface and Sub -Surface Jacket

The need to categorise additions ports that terminate at The bioreactor jacket area is specified with the following
liquid surface and those that are subsurface is determined by considerations in mind :
the operational scenarios and effects of feed strategy on 35 Steam sterilisation at 121 - 125° C .
process control. Warming up of 1914 -3077 liters of medium from 10° C .
The 4000 liter bioreactor has been designed to accept two to 36 .5° C . in < 2 h .
subsurface feeds and alkali that need to be discharged in All points within the bioreactor must reach + 0 .2° C .of set
well-mixed area of the bioreactor. The foam is controlled by point, typically 36 .5° C . as measured by thermo
surface addition of 1 in 10 diluted C - emulsion . A single 40 couples.
spare above surface addition port directed to the vessel wall Chilling of 1914 -3077 liters ofmedium from 36 + 2° C . to
is also designed for future flexibility. The splashing of 10° C . in 2 h .
culture onto the surface of the medium during inoculation of Bioreactor pH Control
the seed bioreactor can be avoided to prevent build up of The process pH is monitored and controlled with probes
foam . Therefore the inoculum addition port is above surface 45 connected via a transmitter to a DCS based process control
and directed to the vessel wall . The use of the harvest port ler. The process pH is controlled by addition of CO2 through
in the base plate is the ideal port for removal of inoculum the control sparger to bring the pH down to set point and
during transfer of inoculum . Additionally the medium addi- addition of alkali to bring pH up to set point.
tion port is directed to the vessel wall . In summary the total 50 Alkali is added through at least one subsurface port at
addition ports are : centre - line of the bottom impeller . The CO2 will be added
Four surface additions with medium inlet , inoculum inlet via the control sparger .
and a spare small addition directed to the vessel wall Control and backup probes are in the lower port ring at
and addition port for antifoam dropped on to the liquid 531 mm from tank bottom as shown in table 17 .
surface away from the vessel wall. 55 Bioreactor DOT Control
Three subsurface additions for feeds and alkali . Dissolved oxygen is monitored and controlled with pola
Sample Ports rographic DOT probe. The DOT set point maintained by
The sample port design is similar to that specified for the sparging:
20 000 liter bioreactor. Initial N , ballast and /or air on demand .
Volume Measurement 60 Air ballast with air on demand .
The level sensor is able to measure up to 4000 liters with Air ballast with oxygen on demand .
an accuracy + 0 . 5 % of full span . DOT control allows DOT set point to be maintained
Bioreactor Temperature Control through interchangeable use of oxygen or air as demand gas.
The 1914 to 3077 liter ofmedium are brought to operating It is not envisaged that pCO , control is required in the
temperature , typically 36 .5° C . by process control. This is 65 inoculum bioreactor. Control and backup probes are in the
achieved by “ gentle ” heating of the jacket and avoid high lower port ring at 531 mm from tank bottom as shown in
temperature at vessel wall. table 17 .
 US 10 ,179 , 898 B2
37 38
Feed Addition Control compromise value determined from volumetric contingency
The point of addition is 531 mm from tank bottom , in the for foam , plant height and acceptable impeller shaft length .
plane of the centre - line of the lower impeller to assist in the The head to base tangent line height is 2 ,347 m .
rapid dissipation of feed bolus . 5 Freeboard Height
Antifoam Addition Control
The addition point is at surface projecting 0 .25xT toward The freeboard height of 500 mm (293 liters or 31 % v /v of
the tank centre or 407 mm from centre of tank . the maximum operating volume) is used for this seed
bioreactor.
10
EXAMPLE 3 : 1000 LITER BIOREACTOR Head and Base Plate
SPECIFICATION
The base and head plate design is ASME F & D for this
Vessel Geometry seed bioreactor.
The vessel geometry for the 1000 liter bioreactor was
determined by an iterative design basis in which the maxi- 15 Bioreactor Agitation Requirement
mum working volume, freeboard straight side distance , Agitation of the bioreactor is to achieve rapid mixing ,
aspect ratio (H / T ) and impeller to tank diameter ratio (D / T ) maintain homogeneity , maintain mammalian cells in sus
are altered until an acceptable aspect ratio is achieved .
Bioreactor Aspect Ratio H , / T pension and gas bubble dispersion . The underlying issue
Table 18 below describes the aspect ratios in the 1000 liter 20 with achieving the above objectives is to minimise cell
bioreactor at various operating volumes during normal pro - damage through shear forces originating from impeller
cessing. These aspect ratios arise from the selection of tank geometry and " eddies” or vortices created behind the impel
ID and the operating volume required . From a processing ler blades. A compromise of the above objectives was
perspective the mixing requirements at the different operat 25 achieved by selection of an appropriate impeller type and
ing conditions are different. During pre -inoculation stage the 25
bioreactor mixing is important to allow medium to equili gassing strategy .
brate with minimal K , a requirement. However with post
inoculation and pre -transfer stages both mixing and Kya are Bottom Versus Top Driven Shaft
important considerations. Therefore both of these features 30 The motor drive is top mounted for the benefits as already
were tested at the aspect ratio range . highlighted
TABLE 18 Baffles
Key operating volumes and aspect ratios The baffle requirement for a centre mounted impeller is
in the 1000 litre bioreactor 35 critical to prevent vortex formation . The critical issues
Liquid Aspect related to baffles are baffle number, baffle width ( W ) , baffle
Volume, L head , mm ratio , H / T length (Hbafe) and baffle to tank wall clearance (W .) .
Stage N -3 Four equally spaced baffles that are 0 .1xT or 86 mm wide
w 1. 1xH - H , or 2099 mm tall and have a baffle to tank wall
Pre -Inoculation
Stage N - 3
250
484
484
0.56
0 . 56
clearance , We of 0 .01xT or 9 mm were used .
The thickness of baffle is not specified but the thickness
Post Inoculation & 300 570 0 .66
needs to ensure rigidity to the radial component of the fluid
Pre -transfer /Harvest
Stage N - 2 flow . Additionally thickness needs to ensure the baffle plates
45 are not warped during SIP thereby affecting the baffle to tank
Pre - Inoculation , 1 . ) 4001 1 . ) 740 1.) 0 . 86
Post-drain Pre 2 .) 50 - 1001 2 . ) 143 - 228 2 .) 0 .17 -0 .26 wall clearance .
refill 3 .) 1922 3 .) 385 3 .) 0 . 45 Impeller Type, Size and Number
Stage N - 2
Post Inoculation & 1 . ) 450 1 . ) 826 1 .) 0 .96
The impellers for the 10001bioreactor should be identical
Pre -transfer/Harvest 2 .) 450 - 9003 2 .) 826 - 1594 2 .) 0 .96 - 1 .84 s formed to the 20 000 liter vessel with an identical D / T ratio .
3 .) 9604 3 .) 1696 3 .) 1. 96 Therefore the bottom impeller is a Lightnin ' s A315 at 381
Pre - inoculation volume and rolling seed inoculation volume for the 1 in 9 sub - cultivation
mm diameter and the top impeller is a Lightnin 's A310 at
process .
Rolling seed inoculation volume for the lin 5 sub - cultivation processes.
381 mm diameter.
Rolling seed post inoculation & pre -transfer volume for the lin 9 sub - cultivation 5
processes .
Rolling seed post inoculation & pre -transfer volume for the lin 5 sub -cultivation
processes.
The impeller spacing (D ) between the centre- line of the
top impeller and the centre -line of the bottom impeller is
Tank Diameter 2xDbottom ( 762 mm ). The off bottom impeller clearance (D )
The tank diameter is altered to obtain the optimal aspect is 0.4xDbottom (152mm ). This allows the bottom impeller to
ratio H , T . Changes to tank internal diameter are limited by " remain submerged with liquid cover (D ) of 0 .5xD bottom or
acceptable aspect ratio and plant footprint. The tank ID is 190 mm at the lowest post- inoculation volume of 167 liters
0 .864 m . and both impeller submerged at 616 liters with liquid head
Tank Height above the upper impeller, D ., of 0.5xDrop (190 mm ).
The tank height is determined from the maximum oper - 65 Table 19 highlights volumes that will form liquid surfaces
ating volume, aspect ratio H , / T , freeboard straight side or lower liquid cover above the impeller Agitation can be
length , base and top plate design . The final tank height is a modified to avoid foaming at these critical volumes.
 US 10 , 179 ,898 B2
39 40
TABLE 19 Agitation Rate Rpm , P /V and Tip Speed
Table 21 specifies the agitation rate for the 1000 liter
Key operating volumes that cause interaction bioreactor. The bioreactor is agitated at around 20 -260
with impellers and liquid surface W /m ", preferably at 55 - 85 W /m ». The agitation strategy was
Vol developed during the 500 liter pilot fermentations. An agi
Interaction ume, L Potential Operation tation rate of up to 155 + 1 rpm is used as an operational
Submerge top impeller 616 Volume seen during inoculation of range .
with 0.5D A310 liquid 1 in 5 processes and rolling oper TABLE 21
cover ation of the 1 in 9 process
Liquid surface touching Volume seen during inoculation of 10 – Agitation rate for the 1000 L bioreactor
top edge of top impeller 1 in 5 processes and rolling oper
ation of the 1 in 9 process Power per unit
Liquid surface touching 492 Volume seen during inoculation of Agitation rate, rpm volume, W · m -3 Tip Speed , m · s -1
bottom edge of top 1 in 5 processes and rolling oper 10 - 155 0 - 150 3. 1
impeller ation of the 1 in 9 process
Submerge bottom impeller 167 Volume seen during inoculation of 1515 20 - 145 0 - 145 2 .9
with 0 .5D4315 liquid cover 1 in 5 processes and rolling oper When both impellers submerged
ation of the 1 in 9 process 2When bottom impeller submerged
Liquid surface touching 90 Volume seen during rolling oper
top edge of lower impeller ation of the 1 in 9 process
Liquid surface touching 21 Volumes seen during pre -inocula Mechanical Seals Specification
bottom edge of lower
impeller
tion fill of 1 in 5 and 1 in 9
processes.
A double mechanical seal that is condensate lubricated as
described was used .
Bioreactor Aeration Requirement
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 liter bioreactor.
The 1000 1 bioreactor operates at two discrete post - 25 Oxygen will not be required for DOT control. However
inoculation volumes with either the bottom impeller sub oxygen enriched air may be used to facilitate lower gassing
merged during the 1 in 5 processes and 1 in 9 processes or to prevent excess foaming . It is recommended that the
with both impellers submerged during the N -2 phase of the smaller range CA MFC should be used to delivery nitrogen
1 in 5 process and rolling seed operations for both 1 in 5 and 30 for early DOT control and reducing deviant, high levels of
1 in 9 processes . DOT.
TABLE 22
Table 20 shows the liquid cover above the upper and
lower impeller during operation of the 1 in 5 and 1 in 9 Gas flow rate and MFC operating ranges
sub -cultivation processes. During rolling operation of the 1 for the 1000 litre bioreactor
35
in 5 and 1 in 9 processes the liquid cover above the lower Gas Operating range Comments
impeller falls below 0 .5xD . It is therefore important to Head Space
reduce the agitation rate , to avoid surface gas entrainment,
whilst operating at this low volume. At 960 liters a liquid 1. Clean air 1. 0 -50 SLPM 1. Head space purging of CO2 and
O,
cover. (D ) of 2 .05xDtop is obtained. At this level Kya has 40 22. Nita
Nitrogen 2. Utility rated 2. For rapid DOT probe zeroing
been shown not to be adversely affected and bulk blending 3. Helium 3. Utility rated 3. Tank integrity testing
is not an issue.
TABLE 20
Key operating volumes and the liquid cover above
top impeller, Do and bottom impeller, DB
Cylinderical Do , DBO Do as ratio DB , as ratio
Operating volume, L height, H (mm ) mm mm of DA310 of DA315
Pre -Inoculation , 250 L 334 332 0.87DA315
Pre -Inoculation , 400 L 590 -- 588 1.54D A315
Post-Inoculation and 419 417 1.10D A315
Pre - Transfer, 300 L
Post-Inoculation and
Pre - Transfer, 450 L
675
675 – 673673 1 .77DA315
Post drain , pre -bulk
192 L
235 233
- 233 0 .61DA315
Post drain , pre -bulk 50
100 L
00 --7878 - 76 76 0 .2D4315
Post- Inoculation and 1443 - 1.78D4310
Pre- Transfer, 900 L
Post - Inoculation and 1545 782 - 2.05D4310
Pre - Transfer , 960 L
Woff bottom impeller clearance, Dc = 152 mm (0 .4D 4315 ), Impeller separation , Ds = 762 mm (2D4315 ), tank
D of 864 mm and Height of ASME F & D base plate, H = 151 mm
( ) Do = H - Ds – (Dc - Hy) and DBQ = H - (Dc - HK )
 US 10 ,179 ,898 B2
41
TABLE 22-continued allows the placement of control sparger at a distance of 88
Gas flow rate and MFC operating ranges
mm ( D . - S .) below the bottom edge of the bottom impeller
for the 1000 litre bioreactor and no greater than 64 mm from tank bottom (S .).
Gas Operating range Comments TABLE 23
Control Sparger Design specification for 1000 litre bioreactor spargers
1. Clean air ? 1 . 2 - 20SLPM 1. Gas flow under DOT control Parameter Control Sparger
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 10 Gas flow , SLPM 35
4 . Nitrogen ? 4 . 0 . 2 -5SLPM 4. Early DOT control by ballast Number of sparge holes 30
5 . Helium 5. Utility rated 5. Tank integrity testing Orifice diameter, d ., m 0 .002
Gas flow , m3 . s ? 5 . 83E - 04
The air and nitrogen gas flow into bioreactor via a bypass for post SIP tank pressurisation, Orifice area, m ? 3 . 14E - 06
Nitrogen delivered via the 0 to 5SLPM CA MFC , could be used during early DOT control. Total orifice area , m ? 9 . 42E - 05
15 Density of air , Kg . m -3 1. 166
The calculation of hole size and number is iterated until Viscosity, Nm : s -2 1 . 85E - 05
the target Reynolds number of gas emerging from holes is Sauter mean diameter, mm 10 .65
< 2000 and the Sauter mean bubble diameter for a bubble (dus = 1.17 V . 0.4 d.0.8 g -0.2)
chain regime is approximately 10 mm . Gravitional acceleration, g , g m · s - 2 9 . 807
for 2020 Density
Table 23 shows the key sparger design specification lor difference, Kg . m - 3
Reynold 's number, > 2000 jetting regime
1048 .834
782
the 1000 liter bioreactor. The sparger length , S , of 305 mm Gas velocity at sparger, V ., m /s 6 . 19
is determined for pipe geometry. The holes are distributed on Sparger length , S?, m 0 . 305
either end of the sparger to preventbubble liberating directly Combined length to drill required holes, m 0 . 06
under the A315 hub . Alternatively a crescent geometry can Number of rows to fit required holes in length Sz , m
be considered . Sparger to tank bottom clearance , Sc , m 0 . 064
The pipe diameter is 25 mm . 30 2 mm holes are located 25 Sparger to bottom impeller clearance , Dc- Sc, m 0 .088
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. Position of Probes , Addition and Sample Ports
The bioreactor port for sparger installation is designed to The design basis for positioning of probes , addition and
fit pipe design of diameter of 25 mm . The position of port sample ports is the same as for the 20 000 1 bioreactor.
TABLE 24
Probe, addition and sampling port specification for the 1000 litre bioreactor

Diameter, mm 'Position , mm
Probe/ Port Location (inches ) ( inches) Rational
Temperature Lower 38 . 1 (1 .5 " ) 1 .) 286 ( 11" ) Positioned to minimise
(main ) ring 2 .) 30° monitored volume
Temperature Lower 38.1 (1.5") 1.) 286 (11" ) Positioned to minimise
(backup ) ring 2 .) 170° monitored volume
PH (main ) Lower 38. 1 (1.5") 1.) 286 (11") Positioned to minimise
ring 2 .) 10° monitored volume
PH (backup ) Lower 38 .1 ( 1.5" ) 1.) 286 ( 11") Positioned to minimise
ring 2 .) 20° monitored volume
DOT (main ) Lower 25 .0 (0. 98 ") 1.) 286 ( 11" ) Positioned to minimise
ring 2 .) 150° monitored volume
DOT (backup ) Lower 25 .0 (0 . 98 ") 1.) 286 (11" ) Positioned to minimise
ring 2 .) 160° monitored volume
Spare - 2 (spare -
pCO2)
Lower
ring
38.1 (1.5") 1.) 286 (11" ) Positioned to minimise
2.) 180° monitored volume
Spare - 3 ( spare - Lower 50 .8 (2" ) 1 .) 286 ( 11 " ) Positioned to minimise
Biomass ) ring 2 .) 1900 monitored volume
Sample valve Lower 38 .1 (1.5 ") 1.) 286 (11" ) NovAseptic type
(main ) ring 2 .) 40°
Sample valve Lower 38. 1 ( 1.5" ) 1.) 286 (11" ) NovAseptic type
(back up ) ring 2 .) 40°
Alkali addition Lower 12 .7 (0 .5 ") 1 .) 286 ( 11" ) Diametrically opposite
ring 2 .) 190° pH probes
Feed 1 Lower 12.7 (0.5 ") 1.) 286 ( 11" ) Diametrically opposite
ring 2 .) 200° pH probes
Feed 2 Lower 12.7 (0 .5") 1.) 286 (11") Diametrically opposite
ring 2.) 210° pH probes
Antifoam addition Head 50 .8 (2" ) 1.) N / A Liquid surface /0 .25T
plate 2 .) 170° from tank centre
Spare surface Head 50.8 (2") 1.) N / A surface directed
addition plate 2 .) 180° to vessel wall
DOT control sparg N / A 50 .8 (2" ) 1.) 64 (2 . 5 ")
er orifice 2 .) 0°
Overlay gas Head 38.1 (1.5") 21..)) N135°
/A Diametrically opposite
plate vent out
Exhaust vent out Head 38.1 (1.5") 1.) N / A Diametrically opposite
plate 2 .) 315° overlay gas in
 US 10 , 179 , 898 B2
43 44
TABLE 24 - continued
Probe . addition and sampling port specification for the 1000 litre bioreactor

Diameter, mm Position , mm
Probe/ Port Location (inches) ( inches) Rational
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° wall
Inoculum transfer Head 50.8 (2 .0 ") 1.) N / A Directed into vessel
from S200 to plate 2 .) 320° wall
1000 L
CIP - Spray ball Head 76 .2 (3 " ) 1. ) N / A CIP ’ing of highest
plate 2 .) 270° point
CIP - Spray ball Head 76 .2 (3 " ) 1.) N / A
plate 2 .) 60°
Pressure gauge Head 38.1 (1.5" ) 1.) N / A
plate 2 .) 500
Rupture disc Head 50. 8 (2 " ) 1. ) N / A
plate 2 .) 280°
Spare nozzle Head 101.6 (4" ) 1 .) N / A
plate 2 .) 160°
1 . Hand hole Head 1. 203 .2 (8 " ) 1.) N / A Single port permitting
2 . Sight glass plate 2 . 101.6 (4 " ) 2 .) 70° two functions
Agitator shaft Head 152.4 (6 " ) 1. ) N / A Centre of head plate
opening plate 2 .) 75°
Measured from the tangential line of the base plate . Degree pertains to plane of clockwise rotation .
(2 ) Diameter of nozzle at the bioreactor

In order to monitor, control and sample from a volume of Bioreactor Temperature Control
501, the probes and port ring needs to be 151 mm from tank The 250 to 800 liters ofmedium is brought to operating
bottom . However the probe/port ring cannot be located this zo temperature , typically 36 .5° C . during initial inoculation and
low as it falls on the weld of the base plate and the straight “ seed rolling operation ” by process control. This is achieved
cylindrical side of the bioreactor. The probe and port ring has by “ gentle” heating of the jacket and avoid high temperature
been specified at 286 mm from tank bottom . This permits a at vessel wall .
volume of 134 liters to be monitored , controlled and Lacket
sampled . The probes/port ring is located asas close
close toto the
the tank
tank z35 The bioreactor jacket area is specified with the following
bottom as permitted to minimise the monitored /controlled considerations in mind :
volume. Steam sterilisation at 121- 125° C .
Addition Ports , Surface and Sub -Surface Warming up of 250 -800 liters of medium from 10° C . to
The 1000 liter bioreactor has been designed to accept two 36 .5º C . in < 2 hrs .
subsurface feeds and alkali to be discharged into a well- 40 All points within the bioreactor must reach 20 . 2° C . of set
mixed area of the bioreactor. The foam is controlled by point, typically 36 .5° C . as measured by thermo
surface addition of 1 in 10 diluted C - emulsion . A single couples.
above surface spare addition port directed to the vessel wall Chilling of 400 liters of medium from 36 + 2° C . to 10° C .
was also integrated for future flexibility . The splashing of in 2 hrs .
culture on to the surface of the medium during inoculation 45 Bioreactor pH Control
of seed bioreactor should be avoided to prevent build up of The process pH is monitored and controlled with probes
foam . Therefore the inoculum addition port is above surface connected via a transmitter to a DCS based process control
and directed to the vessel wall . The use of the harvest port ler. The process pH is controlled by addition of CO2 to bring
in the base plate is the ideal port for removal of inoculum the pH down to set point and addition of alkali to bring pH
during transfer of inoculum . Additionally the medium addi- 50 up to set point. Alkali is added through at least one subsur
tion port is directed on to the vessel wall . In summary the face port at centre-line of the bottom impeller. The CO , is
total addition ports are: added via the control sparger .
Four surface additions with medium inlet, inoculum inlet at 286The control and back up probes are in the lower port ring
and a spare small addition directed to the vessel wall mm from tank bottom to minimise the volume that
and addition port for antifoam dropped on to the liquid 55 Bioreactor
can be monitored as shown in Table 24 .
DOT Control
surface away from the vessel wall. Dissolved oxygen is monitored and controlled with pola
Three subsurface additions for feeds and alkali. rographic DOT probe . The DOT set point maintained by
Sample Ports sparging : -
ne 6060 Initial N2 ballast and /or air on demand
The sample port design is similar to that specified for the
20 000 liter bioreactor. The sample port is located 286 mm Air ballast with air on demand
from tank bottom to minimise the volume that can be Air ballast with oxygen on demand
sampled . DOT control allows DOT set point to be maintained
through interchangeable use of oxygen or air as demand gas .
Volume Measurement 65 Control and back up probes are in the lower port ring at
The level sensor is able to measure up to 1000 liters . The 286 mm from tank bottom minimise the volume that can be
level sensor sensitivity is at least 0 . 25 % of full span . monitored , as shown in table 24 .
 US 10 , 179 ,898 B2
45 46
Feed Addition Control of a batch . Table 26 shows the pre -inoculation volume,
The point of addition is 286 mm from tank bottom , in the inoculation volume and transfer or harvest volume for each
vicinity of the centre-line of the bottom impeller to assist in of the three inoculum expansion stages and the production
the rapid dissipation of feed bolus. bioreactor.
Antifoam Addition Control The seed bioreactors ( stage N - 1 to N - 3 ) are unlikely to be
The addition point is at surface projecting 0 .25xT toward fed therefore the maximum operating volume will be at
the tank centre or 216 mm from tank centre . inoculation . The operating volume range for the 4000 liter
seed bioreactor (stage N - 1 ) is 1914 to 3846 liters . In order
EXAMPLE 4 : BIOREACTOR TRAIN to design a bioreactor that can grow cells from 20 % v / v seed
10 split ratio , the 1000 liter seed bioreactor (stages N - 2 and
The bioreactor design is based on the ability to perform N -3 ) will operate at two operating ranges. For the 11 % v /v
both 1 in 5 ( 20 % v / v ) and 1 in 9 ( 11 % v /v ) subculture ratios . seed split ratio the bioreactor train can produce sufficient
The bioreactor train consists of a 1000 liter (Stages N -3 and culture to meet forward processing cell concentration crite
ria in a single expansion /sub - cultivation stage . However the
N -2) and 4000 liter (Stage N -1 ) seed bioreactors followed by 15 bioreactor
a 20 000 liter production bioreactor (Stage N ). The operating stages to meet train requires two expansion /sub -cultivation
volumes for each bioreactor are defined in examples 1 to 3 . split forward processing criteria for 20 % v /v seed
The bioreactors are based on a stirred tank design and a top
ratio process . Thus for 11 % v / v seed split ratio process
driven agitator system was used . an operating range of 400 to 450 liters is required and for the
20 % v /v seed split ratio process an operating volume range
The design is based on the need to ensure a homogenous 20 of 250 to 960 liters is required .
environment with respect to process parameters such as pH ,
dissolved oxygen tension (DOT) and temperature , maintain TABLE 26
ing a well mixed cell suspension and blending nutrient feeds
within the bioreactor. This provides the necessary physico Vessel sizes for bioreactor train
chemical environment for optimal cell growth , product 35 1000 4000 20000
accumulation and product quality . Key to the design phi litre litre litre
losophy is the need to maintain geometric similarity . This
allows a scale down model to be developed at 12 liter Stage N -3 N -2 N -1 N
laboratory and 500 liter pilot scales. The design of the seed
and production bioreactors are based on the same principles 30, 11V /v% production
v /v Seed with 4 to 25 %
feed
although some departures are required to allow for flexibility
in processing. The aspect ratios (HLT ) selected are typical of Pre -inoculation Volume (L ) 400 - 1914 15456
those used in mammalian cell culture and are in the range 17096
0 . 17 to 1 . 96 post-inoculation . Inoculation Volume (L ) 2153 17391
19231
35 Transfer or Harvest 450 - 2153 20000
TABLE 25 Volume (L ) 21739
20 % v /v Seed with 4 to 25 %
Key bioreactor design parameters V / v production feed
1000 litre 4000 litre 20000 litre Pre -inoculation Volume ( L ) 250 768 2782 13913
40 Inoculation Volume (L ) 3077 15385
Aspect ratio H( T ) 0 . 17 - 1 . 96 0 .63 - 1 . 21 0 . 83 - 1 . 34 300 960 3478 17391
Impeller to tank 0 .44 - 0 . 46 0 .44 - 0 . 46 0 .44 - 0 . 46 3846 19231
diameter ( D / T ) Transfer or Harvest 300 960 3478 20000
Operating Volume 50 -960 1914 - 3846 13913 -21739 Volume (L ) 3846 21739
(L ) Assumed operating volume
Agitator speed 0 -155 0 - 88 0 - 80
( rpm ) 45 Minimum Volume (L ) 250 1914 13913
Control sparger 2 - 20 0 -60 0 - 1000 Maximum Volume ( L ) 960 3846 21739
CA (SLPM ) Ratio of Maximum volume/ 3 . 84 2 . 01 1.56
Ballast sparger No ballast sparger No ballast sparger 0 - 500 Minimum volume
CA /N , flow
(SLPM )
Cultivation resi 2 -51 2 -5 10 - 15 50 It is recommended that the 1000 liter seed bioreactor is
dence time (days) inoculated from culture produced in an S200 Wave biore
Feed additions 2 surface 2 surface 4 surface actor .
3 sub -surface 3 sub -surface 4 sub-surface
1000 1: This bioreactor is operated in batches of up to 5
The culture residence time in 1000 litre bioreactormay be higher depending on the length
of time the bioreactor is repeatedly sub -cultivated or " rolled ” .
days, with potential “ shot additions” of feeds, for cultivation
55 of mammalian cells . However due to repeated drain and
The design constraint is based upon a seeding ratio of refill operation at the end of each batch the total process
11% v /v (1 in 9 dilution ) and 20 % v /v (1 in 5 dilution ), with residence time in this bioreactor can exceed 30 days. The
feed application of 4 % v / v to 25 % v /v of the post-inocula - mammalian cells are kept in a homogeneous suspension by
tion volume. The post- inoculation volume in the production agitation via an identical impeller system to the 20 000 liter
bioreactor is adjusted for feed applications up to 15 % such 60 bioreactor. Additionally other features will be kept geometri
that after the addition of all the feeds the final volume at cally similar to the 20 000 liter bioreactor, where possible .
harvest ends up at 20 000 1. However for feed applications Sparging air or oxygen and air or nitrogen respectively
greater then 15 % v /v the post- inoculation volume is adjusted will control process DOT. Process pH is controlled by
for a 15 % v /v feed but following the application of feeds the addition of alkali for base control and of sparged CO2 for
final pre -harvest volume will be a minimum of 20 000 and 65 acid control.
a maximum 22 000 liters. The production bioreactor is The process operating volumeof the bioreactor changes at
expected to hold a total of 20 000 to 22 000 liters at the end different phases of operation . Initially the bioreactor is
 US 10 , 179,898 B2
47 48
aseptically filled with a bolus ofmedium at 250 to 400 liters the sparger type used , power per unit volume imparted by
in 0 .5 h . The bioreactor is operated in a pre -inoculation impellers and superficial gas velocity of sparged gasses .
phase to bring the process variables to predefined set points . 20 000 1: This bioreactor is operated in batch or fed batch
50 liter culture from a (N -4 ) S -200 seed wave bioreactor is mode for 10 to 15 days for the cultivation of mammalian
inoculated , by pneumatic assisted flow , or pumped with a 5 cells . The mammalian cells are kept in a homogeneous
peristaltic pump in 25 to 30 minutes into the 1000 liter suspension by agitation via an impeller system .
The process operating volume of the bioreactor changes at
bioreactor at 1 in 5 or 1 in 9 dilutions. The post- inoculation die
operating volume is 300 and 450 liters for 1 in 5 and 1 in 9 different phases of operation . Initially the bioreactor is
seeded process respectively . The addition of alkali for base aseptically filled with cell culture medium at 13913 to 17096
control and 1 in 10 antifoam suspension for suppression of 10 liters in 1 - 2 h . The bioreactor is operated in a pre - inoculation
phase to bring the process variables to predefined set points .
foam contributes towards the final volume. The inoculum Culture from the 4000 liter seed bioreactor (N - 1 ) is inocu
culture may be fed by a “ shot addition ” if the culture interval lated by pneumatic flow at a flow rate range of < 4000 1/h into
is longer then expected . As a result ofmixing and gassing the the 20 000 liter bioreactor at 1 in 5 or 1 in 9 dilutions. The
liquid volumes described above will expand due to gas hold 15 post- inoculation volume continuously increases following
up . The extent of this rise is dependent on the sparger type an application of sub - surface feeds to maximum of 20 000
used , power per unit volume imparted by impellers and liters (two feeds totalling 4 to 25 % v / v ). The addition of
superficial gas velocity of sparged gasses. alkali for base control and 1 in 10 antifoam suspension for
The N -3 stage ends when viable cell concentration suppression of foam accounts for about 100 liters and 20
reaches transfer criteria. The N - 2 stage for 1 in 5 process 20 liters respectively . As a result of mixing and gassing the
begins with a bulk up in volume to 960 liter by draining of liquid volume expands due to gas hold up . The extentof this
192 liter excess culture and addition of 768 liter fresh rise is depended on the sparger type used ( fluted or sintered),
medium in 1 . 5 h . 696 to 769 liter of culture are transferred power per unit volume imparted by impellers and superficial
at the end of N - 2 stage to the 4000 liter bioreactor for the 1 gas velocity of sparged gasses.
in 5 processes. For the 1 in 9 processes 239 liters are 25 Table 27 describes the aspect ratios in the 20 000 liter
transferred to the 4000 liter bioreactor. bioreactor at various operating volumes during normal pro
The 1000 1 bioreactor is continuously " drained and cessing. The aspect ratios have been tested at 500 liter scale
refilled with fresh medium ” or “ rolled ” to provide back up and provided the superficial gas velocity and power per unit
culture for the 4000 liter bioreactor. The duration of the volume are kept constant the Kya remains constant.
rolling seed operation is dependent on the length of the 30
production campaign and the permissible elapsed genera TABLE 27
tions number of the seed culture . Typically it is assumed that
rolling seed operation is in excess of 30 days. The rolling Key operating volumes and aspect ratios
operation consists of retaining approximately 192 liters of in the 20 000 litre bioreactor
the 960 liter culture and diluting with 768 liter fresh medium 35 Liquid Aspect
for the 1 in 5 processes . For the 1 in 9 processes the 1000 Volume, L head , mm ratio , H / T
liter bioreactor is expected to be " rolled ” by retaining 50 to Pre- Inoculation 13913 - 17096 2458 - 2977 0 . 88 - 1.07
100 liter of the 450 to 900 liter culture and diluting with 400 Post Inoculation 17391 - 19231 3025 - 3325 1 .08 - 1 . 19
to 800 liter fresh medium . Process control ranges are relaxed Harvest 20000 - 21739 3451 - 3734 1 . 23 - 1 .34
over this operation . The medium added to the bioreactor 40 -
during rolling operation is warmed to 30° C .
4000 1: This bioreactor is operated in batch of no more What is claimed is:
then 5 days , with potential “ shot additions” of feeds, for 1 . A scalable bioreactor for the cultivation of mammalian
cultivation of mammalian cells. The mammalian cells are cells, the bioreactor comprising:
kept in a homogeneous suspension by agitation via an 45 a biocompatible tank or vessel having a volume of
identical impeller system described in example 1 . Addition between 500 liters and 20 , 000 liters and at least one top
ally this vessel is geometrically similar to the 20 000 liter impeller and at least one bottom impeller ,
bioreactor. wherein the at least one top impeller has a power number
Sparging air or oxygen and air or nitrogen respectively ( N ) between 0 . 1 and 0 . 9 ,
controls process DOT. Process pH is controlled by addition 50 wherein the at least one top impeller has a flow number
of alkali for base control and of sparged CO2 for acid ( N . ) between 0 .4 and 0 . 9 ,
control. wherein the at least one bottom impeller has a power
The process operating volume of the bioreactor changes at number (N ) between 0 .5 and 0 . 9 ,
different phases of operation . Initially the bioreactor is wherein an impeller spacing (Ds) between the at least one
aseptically filled with a bolus ofprotein free medium at 1914 55 top impeller and the at least one bottom impeller is
to 3077 liters in 1.5 h . The bioreactor operates in a pre between 1.229xthe diameter of the bottom impeller
inoculation phase to bring the process variables to pre (Dbottom ) and 2xD bottom
defined set points. Culture from the 1000 liter (N - 2 ) seed wherein the height of a liquid above the at least one top
seed bioreactor is inoculated by pneumatic flow at a flowrate impeller ( D ) is between 0 .3xthe diameter of the at
to allow transfer in one hour, at 1 in 5 or 1 in 9 dilutions. The 60 least one top impeller (Diop) and 2 .5xDtop, and
post- inoculation operating volume is 2153 to 3846 liters. wherein a bottom clearance (D ) between the tank bottom
The addition of alkali for base control and 1 in 10 antifoam and the center - line of the bottom impeller is at least
suspension for suppression of foam contributes towards the 0 . 35xDbottom
final volume. The inoculum culture may be fed by a “ shot 2 . The bioreactor according to claim 1, wherein the at
addition ” if the culture interval is longer then expected . As 65 least one top impeller has the power number (N .) between
a result of mixing and gassing the liquid volume expands 0 . 25 and 0 . 35 and the at least one bottom impeller has the
due to gas hold up . The extent of this rise is dependent on power number (N . ) between 0 .70 and 0 .80 .
 US 10 , 179 ,898 B2
49 50
3 . The bioreactor according to claim 1, wherein the at 12 . The bioreactor according claim 9 , wherein the biore
least one top impeller has the power number (N ) of 0 .30 and actor has at least one baffle and the bioreactor has a length
the at least one bottom impeller has the power number (N . ) of the at least one baffle at 1.1xa total straight height of the
of 0 . 75 . bioreactor (H ), wherein the bioreactor has a width of the at
4 . The bioreactor according to claim 1 , wherein at least 5 least one baffle at 0 . 1xthe internal diameter of the tank, and
one bottom impeller has a flow number (N .) between 0.50 wherein the bioreactor has a height of the at least one baffle
and 0 .85 .
5 . The bioreactor according to claim 1, wherein the ((Hhafte H ) - a
) at 1. 1xthe total straight height of the bioreactor
head height of the bioreactor (H1).
impeller to tank diameter ratio is between 0 . 35 and 0 .55 .
13 . The bioreactor
6 . The bioreactor according to claim 1, wherein the 10 or vessel has a volume according to claim 1 , wherein the tank
impeller to tank diameter ratio is between 0 . 40 and 0 .48 . of 10 ,000 liters .
7 . The bioreactor according to claim 1 , wherein the tank 14 . The bioreactor according to claim 1, wherein the tank
or vessel has a volume of 500 liters . or vessel has a volume of 20 ,000 liters .
8 . The bioreactor according to claim 1 , wherein the tank 15 . The bioreactor according to claim 14 , wherein the
or vessel has a volume of 1000 liters. 15 bioreactor has at least one sparger, wherein a clearance (Se)
9 . The bioreactor according to claim 1 , wherein the tank between the at least one sparger to the tank bottom is
or vessel has a volume of 4000 liters . between 560 mm and 620 mm , and wherein a clearance
10 . The bioreactor according claim 9 wherein the biore - (D - S .) between the at least one sparger to the center- line of
actor has at least one sparger, wherein a clearance (S .) the bottom impeller is between 300 mm and 340 mm .
16 . The bioreactor according to claim 1 , wherein the
between the at least one sparger to the tank bottom is at least 20 bioreactor
0 . 17xsparger length (S ), and wherein a clearance ( D . - S .) maintains a homogeneous environment such that :
between the at least one sparger to the center- line of the a ) pH is maintained within + / - 0 .03 of a pH set point;
bottom impeller is 0 .25xsparger length ( SL ). b ) the dissolved oxygen tension (DOT) is maintained with
11 . The bioreactor according claim 9 , wherein the biore + / - 2 % of a DOT set point; and
actor has at least one sparger, wherein a clearance (Se) 25 c) temperature is maintained within + / -0 .2° C . of a
between the at least one sparger to the tank bottom is temperature set point.
between 315 mm and 360 mm , and wherein a clearance 17 . The bioreactor according to claim 16 , wherein the
( D - S .) between the at least one sparger to the center- line of temperature set point is from 36 to 38° C .
the bottom impeller is between 180 mm and 205 mm . * * * *