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Klckner et al. Journal of Biological Engineering 2013, 7:28

http:// .!"ioleng.org/content/7/1/28

#E$E%#&' (pen%cce))

&orrelation"eteen *a)) tran)fer coefficient k+aan rele-ant operating para*eter) in clinricali)po)a"le )haken "ioreactor) on a "enchtopilot)caleolf Klckner

1,#ia ace*

1,i"or %nerlei

2,icole #a-en

3,$tefan $chill"erg

3,&le*en) +atter*ann

1

an Jochen B4ch)15

%")tract

Backgroun: %*ong i)po)a"le "ioreactor ))te*), clinrical or"itall )haken "ioreactor) )ho i*portant

a-antage). hepro-ie a ellefine hrona*ic flo co*"ine ith e6cellent *i6ing an o6gen tran)fer

for *a**alian an plant cell culti-ation). $ince there i) no knon uni-er)al correlation "eteen the -olu*etric

*a)) tran)fer coefficient for o6gen k+a an rele-ant operating para*eter) in )uch "ioreactor ))te*), the ai* of

thi) current )tu i) to e6peri*entall eter*ine a uni-er)al k+a correlation.

#e)ult): % #e)piration %cti-it onitoring $)te* #%($9 a) u)e to *ea)ure k+a -alue) in clinrical i)po)a"le

)haken "ioreactor) an Buckingha* :);heore* a) applie to efine a i*en)ionle)) e2 cell )u)pen)ion culture).

&onclu)ion: he re)ulting e

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Cage 2 of 1J

)

$

1

g2

g

3

e)pite the increa)ing u)age of i)po)a"le culti-ation

))te*), onl fe )cientific pu"lication) ha-e reporte

a"out their characteriNation ith re)pect to poer input,

o6gen tran)fer an *i6ing perfor*ance 8F. B con

tra)t, )tirre )tainle)) )teel "ioreactor) ha-e "een e6ten

)i-el characteriNe an opti*iNe o-er the pa)t M0 ear)

-aria"le) for k+a in clinrical or"itall )haken "io

reactor) an their corre)poning unit) are the -olu

*etric *a)) tran)fer coefficient for o6gen k+a1

, the reactor ia*eter *F, the )haking ia*eter 0

*F,

the )haking fre

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Cage 3 of 1J

alilei nu*"er

3

Q2

$ch*itnu*"er Q(2

hi) )et of i*en)ionle)) nu*"er) a) al)o propo)e

" 'enNler an $cheel 2MF to e-elop a )cale

inepenent correlation for the *a)) tran)fer coefficient

in )hake fla)k). he applie nu*"er) for )urface aerate

"ioreactor) iffer fro* i*en)ionle)) nu*"er) that are

co**onl u)e to e)cri"e the *a)) tran)fer in "u""le

aerate "ioreactor) "ecau)e the principle of *a)) tran)

fer iffer) funa*entall in "oth ))te*). =n contra)t to

)urface aerate or"itall )haken "ioreactor), *a)) tran)

fer in "u""le aerate "ioreactor) epen) on the

a*ount, )iNe, "reakup an coale)cence of ga) "u""le).

he characteri)tic) of ga) "u""le) are )trongl influ

ence " the -olu*etric poer input, hich i) therefore

co**onl u)e a) a para*eter for k+a correlation) for

"u""le aerate "ioreactor). he)e a)pect) are not rele

-ant for the utiliNe )urface aerate "ioreactor) that are

operate ithout "u""le aeration.he nu*"er) are inepenent fro* one another an

the )et of nu*"er) can "e tran)for*e into a ifferent

)et of i*en)ionle)) nu*"er), ith no influence on the

re)ulting e6ponent) of the inclue -aria"le), a) long a)

the rule) of Buckingha*) ; heore* are pre)er-e

2IF. =n thi) )et of i*en)ionle)) nu*"er) the Kroue

nu*"er i) u)e to e6pre)) the ratio of centrifugal to

gra-itational force) uring )haking. he relati-e filling

-olu*e i) con)iere ith the Dolu*e nu*"er an the

eo*etric nu*"er i) u)e to e)cri"e the ratio of )hak

ing ia*eter to reactor ia*eter. he height an )hape

of the free li

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1

e)cri"e " the $ch*it nu*"er. =n "ioreactor) ith

out "u""le aeration, the influence of the )urface ten)ion

on the *a)) tran)fer coefficient ecrea)e) ith increa)

ing reactor )iNe a) alrea e)cri"e " oig et al. 27F

an 'er*ann et al. 28F for *icrotiter plate). &on)e

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igure 1 k+a -alue) of 10 +, 20 + an I0 +"ioreactor) at 2I\& eter*ine ith #%($ u)ing 1 a2$(3 )olution ith 107

&o$(J,

0.012 pho)phate"uffer, initial p' [ 8, ata point) connecte ith tren line) for relati-e filling -olu*e) of 2IA, I0A an 7IA.

ofpha)e operation uring the *ea)ure*ent) in the

pre)ent ork. he final range of applica"ilit for the

e6ponent U of the eo*etric nu*"er a) efine "e

teen 0.0M an 0.2.

De))el) of ifferent )iNe) ere u)e to -ar the reactor

ia*eter an, in thi) a, the alilei nu*"er. he influ

ence of the Dolu*e nu*"er an the eo*etric nu*"er

ere con)iere ith their re)pecti-e e6ponent) an

*ultiplie ith the k+a nu*"er a) )tate in igure I. %n

a-erage e6ponent of V [ 0.12 a) eter*ine ith a -al

iit range for the alilei nu*"er of "eteen M.2

10

10

an 1.881012

.

he -ariation of the $ch*it nu*"er in E

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1

igure 3 k+a nu*"er a) a function of the Dolu*e nu*"er for 10 an 20 +"ioreactor) at 2I\&, relati-e filling -olu*e -arie "eteen

2IA an 7IA at )haking fre

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#%($ u)ing 1 a2$(3 )olution ith 107

&o$(J,0.012 pho)phate "uffer, initial p' [ 8, poer function) ith a-erage

e6ponent U [ 1.0M fitte to ata point).

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0 YIZ

igure I Crouct of k+a nu*"er, Dolu*e nu*"er an eo*etric nu*"er a) a function of the alilei nu*"er *ea)ure at 2I\&,

reactor ia*eter -arie "eteen 0.2I c* an 0.7I c* u)ing 10 +, 20 +, I0 + an 200 +"ioreactor), k+a -alue) eter*ine ith

#%($ u)ing 1 a2$(3 )olution ith 107

&o$(J,0.012 pho)phate "uffer, initial p' [ 8, poer function) fitte to ata point)for ifferent )haking fre

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1 *ol/+ a2$(3 0.IM1 1.M88 ` 10L

1.8 ` 10M

1 ` 107

*ol/+ &o$(

0.012 *ol/+ pho)phate "uffer p' 89

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igure M Crouct of k+a nu*"er an alilei nu*"er a) a function of the $ch*it nu*"er $c9 *ea)ure ith 20 +"ioreactor) at 2I\&

u)ing ater $c [ 1M9 an )ulfite )olution) ith 0.I $c [ I809 an 1 a2$(3 $c [ 8779 ith 10

7

&o$(J an 0.012 pho)pha te"uffer, initial p' [ 8, poer function) fitte to ata point) for ifferent )haking fre29 )u)pen)ion cell). % *a6i*u* (# -alue of

igure 7 &o*pari)on "eteen *ea)ure an calculate k+a -alue) at 2I\& for &orning roller "ottle) 2 +9,algene -e))el) 10 +, 20 +,

I0 +9 an &ultiBag (#B 200 +9"ioreactor). $haking fre

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+ X

M]8 **ol/+h9 a) *ea)ure uring the culti-ation of

to "acco B>2 cell) in 2I0 *+ )hake fla)k) u)ing

ura)hige an $koog *eiu* )upple*ente ith 30 g/+

)ucro)e 3IF. hu), a *a6i*u* o6gen tran)fer capacit

of (#*a6 [

8 **ol/+h9 i) re2 cell) in or"itall

)haken "ioreactor). %) )pecifie ith E2 cell) in a 20 + "ioreactor ))te*

co*pare to a 2I0 *+ )hake fla)k culture. he i))ol-e

o6gen ten )ion (9 a) aitionall *onitore in the

20 +"ioreac tor ))te* igure L9. he ( )ignal a)

ala) a"o-e

20A uring the culti-ation, inicating a )ufficient o6gen

)uppl. %) epicte in igure L, a co*para"le tren of

groth para*eter)"eteen the 20 + "ioreactor ))te* an

2I0 *+ )hake fla)k) a) o")er-e. %n increa)e of thefilling

-olu*e in the 20 + reactor ))te* fro* I + to 10 + at

otheri)e e2 cell) culti-ate in ura)hige an $koog *eiu*

containing 30 g/+ )ucro)e. he re

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the correct application of clinrical i)po)a"le )haken

"ioreactor) on a "ench to pilot)cale.

aterial) an, *etho,)

&ulti-ation an agitation ))te*)

a"le 2 pre)ent) an o-er-ie of the ifferent geo*

etrie) an *aterial propertie) of the utiliNe i)po)a"le

reactor ))te*) an the re)pecti-e )haking platfor*u)e in thi) )tu.

oel for the ga)/li

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+

+(

+

OxygenTransferRate(OTR)

[mmol/(Lh)]

Osmolality(Osm)

[mOsm/kg]

pHvalue

[-]

resh!eight

(")

[g/L]

#issolve$OxygenTension

(#OT)

[%]

#ry!eight(#")

[g/L]

igure 8 (6gen ran)fer #ate (#9 uring the culti-ation of . ta"acu* B>2 % cell) culti-ate in ura)hige an $koog

*eiu* in a 10 +"ioreactor, D+ [ 3 +, n [ 1M0 rp*, ,0 [ 7 c* an 2I0 *+ )hake fla)k, D+ [ I0 *+, n [ 180 rp*, ,0 [ I c*.

the concentration ifference "eteen o6gen at the )atu he o6gen concentration &

i) a function of the o6

rate ga)/li

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*eiu*P 20 +"ioreactor, D+ [ I +, n [ 1M0 rp*, ,0 [ 7 c* an 2I0 *+ )hake fla)k), D+ [ I0 *+, n [ 180 rp*, ,0 [ I c*.

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aerate )tirre tank reactor) 2F. %) co*parati-el lo

o6gen tran)fer rate) of le)) than 1M **ol/+h9 ereachie-e ith the i)po)a"le reactor ))te*) in the

pre)ent ork, the a))u*ption of &+ 0 *ol/+9 i), in

thi) ca)e, applica"le for the eter*ination of k+a -alue).

$oiu* )ulfite #oth, Karl)ruhe, er*an, purit ^

L8A9 i))ol-e in eioniNe ater an catalNe ith

co"alt )ul fate luka, euGl*, er*an9 a) u)e for

the o6ia tion reaction in the li(C9 fro* Cre$en) Cre

$en) *"', #egen)"urg, er*an9 ere applie to

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igure 11 E6peri*ental )etup of the #%($ for eter*ining k+a -alue) in clinrical i)po)a"le "ioreactor) ith )en)or) for the

o6gen partial pre))ure p(29 an total air pre))ure pa")9integrate in the reactor capP *a)) flo controller, inlet an outlet -al-e)

an )terile filter) u)e for acti-e aeration.

*ea)ure the i))ol-e o6gen ten)ion (9 in the li

2plant cell

)u)pen)ion culture)

or e-aluation of the k+a correlation the tran)genic

. ta"acu* c-. Bright >ello2 B>29 % cell line

proucing the hu*an anti"o 12 a) u)e. he gen

eration of the tran)genic B>2 cell line i) e)cri"e "

#a-en et al. I2F. B>2 plant cell )u)pen)ion culture)

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ere culti-ate at 2M\& in the ark u)ing ura)hige

an $koog *eia I3F ith *ini*al organic) M8LL,

$ig*a %lrich, $aint +oui), (, G$%9 )upple*ente ith

30 g/+ )ucro)e, 0.2 g/+ K'2C(, 0.M *g/+ thia*ine'&l,

0.2 *g/+2.Jichloropheno6acetic aci 2.9 an, here)tate,

aitional 100 **ol/+ K(3. he p' of the culture

*eiu* a) a!u)te to I.8 ith 1 *ol/+ K('

"efore autocla-ing for 21 *in at 121\&. Clant cell

)u)pen)ion) cul ture) ere )u"culti-ate eekl for cell

*aintenance in

2I0 *+ Erlen*eer fla)k), fille ith I0 *l cell )u)pen)ion,

)eale ith a cotton plug an )haken at 180 rp* an

2M\& in the ark. =noculation a) conucte" aing IA

D/D9 of a )e-en a ol culture to fre)h *eiu*.

(6gen tran)fer rate *ea)ure*ent) in )hake fla)k)he (# )ignal) in 2I0 *+ Erlen*eer fla)k) ere

&o*peting intere)t)

he author) eclare that the ha-e no co*peting intere)t).

%uthor)contri"ution)

K conucte e6peri*ent), *ae the ata e-aluation an rafte the

*anu)cript. # an %pro-ie aitional ata an helpe to conuct thee6peri*ent).#pro-ie plant cell )u)pen)ion culture), helpe to conuct

the e6peri*ent) an re-iee the *anu)cript. $$ coorinate the

e6peri*ent) an re-iee the *anu)cript. &+ calculate o6gen iffu)ion

coefficient) an )olu"ilitie) of the applie )olution). JB helpe to e)ign an

coorinate the )tu an re-iee the *anu)cript. %ll author) rea an

appro-e the final *anu)cript.

%cknoleg*ent)he author) thank the co*pan K4hner % for their helpful )upport ith

e, Ceter &C, %kg4n %, B4ch) J: Coer con)u*ption an heat tran)fer

re)i)tance in large rotar )haking -e))el). Bioche* Eng J 200, 21:83]L1.

12. e Je)u) J, irar C, Bourgeoi) , Bau*gartner , Jacko B, %*)tutN ',

ur* : u"e$pin )atellite): a fa)t track approach for proce))

e-elop*ent ith ani*al cell) u)ing )haking technolog. Bioche* Eng J

200, 17:217]223.

13. Rhang , $tettler , e $ancti) , Cerrone , Carolini , i)cacciati , e

Je)u) , 'acker , uarteroni %, ur* : G)e of or"ital )haken i)po)a"le

"ioreactor) for *a**alian cell culture) fro* the *illiliter)cale to the

1,000liter )cale. %- Bioche* Eng/Biotechnol 2010, 11I:33]I3.

1. #a-al K, Kato >, B4ch) J: &o*pari)on of tor

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17. +iu &, 'ong +3: e-elop*ent of a )haking "ioreactor ))te* for

ani*al cell culture). Bioche* Eng J 2001, 7:121]12I.

18. Rhang , B4rki &%, $tettler , e $ancti) , Cerrone , i)cacciati ,

Carolini , eJe)u) , 'acker +, uarteroni %, ur* : Efficient

o6gen tran)fer " )urface aeration in )haken clinrical container)

for *a**alian cell culti-ation at -olu*etric )cale) up to 1000 +.

Bioche* Eng J 200L, I:1]7.1L. erner $, Ei"l #, +etten"auer &, #oll , Ei"l , e Je)u) , Rhang , $tettler

, i))ot $, B4rki &, et al:=nno-ati-e,non)tirre"ioreactor) in)cale)fro*

*illiliter) up to 1000 +iter)for )u)pen)ion culture) of cell) u)ing

i)po)a"le"ag) an container) a )i)) contri"ution.&hi*ia 2010,

M:81L]823.

20. $tettler , Rhang , 'acker +, e Je)u) , ur* : o-el or"ital )hake

"ioreactor) for tran)ient prouction of &'( eri-e =g).

Biotechnol Crogr 2007, 23:130]13M.

21. i))ot $, ichelC(,'acker +, Bali +, e Je)u) , ur* :k+9a a) a

preictor for )ucce))ful pro"einepenent*a**aliancell

"ioproce))e) in or"itall)haken"ioreactor).e Biotechnol2012,

2L:387]3L.

22. i))ot $, ("er"ek %, #eclari , reer , 'acker +, Bali +, arhat , ur*

: Efficient an reprouci"le *a**alian cell "ioproce))e) ithout

pro"e) an controller) e Biotechnol 2011, 28:382]3L0.

23. Rhang , $tettler , #eif (, Kocourek %, eJe)u) , 'acker +, ur* :

$haken helical track"ioreactor): Cro-iing o6gen to highen)it

culture) of *a**alian cell) at -olu*e) up to 1000 +" )urface aeration

ith air. e Biotechnol 2008, 2I:M8]7I.

2. ri*-all : &haracteri)tic , Koh $, +ee >$: i6ing ti*e an poer

con)u*ption for a li

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