Very high cell density perfusion of CHO cells in disposable bioreactor, challenge or reality

Transcription

1 Very high cell density perfusion of CHO cells in disposable bioreactor, challenge or reality Véronique Chotteau Cell Technology group (CETEG) KTH, School of Biotechnology, Div. Bioprocess March 28, 2012 Véronique Chotteau, CETEG, KTH 1

2 Perfusion in industry for animal cell Majority of fed-batch processes world-wise Perfusion used traditionally for unstable proteins in companies or lab s with culture perfusion, i.e. where knowledge, competence and PEOPLE are present Perfusion more technically challenging à higher risk of failure, higher risk of contamination smaller cultivation vessel less process development constant cellular environment is beneficial for cell metabolism and product quality Perfusion equipment robust and disposable robust equipment à higher risk of failure disposable equipment à higher risk of contamination March 28, 2012 Véronique Chotteau, CETEG, KTH 2

3 Three systems studied at CETEG (KTH) Collaborations WAVE Bioreactor equipped with ATF GE Healthcare (Sweden, USA) WAVE Bioreactor equipped with TFF GE Healthcare (Sweden, USA) CellTank CerCell (Denmark), Belach (Sweden) March 28, 2012 Véronique Chotteau, CETEG, KTH 3

4 WAVE Bioreactor in perfusion with ATF or TFF Goal Evaluation of disposable WAVE Bioreactor in perfusion mode Evaluation of two types of cell separation based on hollow fiber filtration: Alternating Tangential Flow filtration Tangential flow filtration Evaluation of the limits of the system Strategy Cell line #1 = IgG producing Chinese Hamster Ovary cells Study of perfusion à learning phase and study of the equipment Study of perfusion à study of the limits of the system Evaluation for application of IgG production and comparison with fed-batch Evaluation for application of cryopreservation / cell banking WAVE Bioreactor source: March 28, 2012 Véronique Chotteau, CETEG, KTH 4

5 CellTank in perfusion mode Goal Evaluation of disposable CellTank in perfusion mode (CerCell, Denmark) Evaluation of the system Strategy Cell line #2 = IgG producing Chinese Hamster Ovary cells Study of perfusion à trouble shooting / learning phase and study of the equipment Study of perfusion à Evaluation of the system at very high cell density Evaluation of effect of temperature decrease March 28, 2012 Véronique Chotteau, CETEG, KTH 5

6 Introduction and System set-up March 28, 2012 Véronique Chotteau, CETEG, KTH 6

7 Perfusion devices connected to WAVE Bioreactor ATF (REFINE Technology) TFF Alternating Tangential Tangential Flow with ReadyToProcess filter Flow Filtration with ReadyToProcess filter GE Healthcare GE Healthcare WAVE Bioreactor TM WAVE Bioreactor TM source: March 28, 2012 Véronique Chotteau, CETEG, KTH 7

8 ATF & WAVE Bioreactor TFF & WAVE Bioreactor 1 L/min (3400 s-1) 1 L/min (3400 s-1) WAVE source: Cellbag 10 L Hollow fiber filter ATF-2 March 28, 2012 Véronique Chotteau, CETEG, KTH TFF Pump Watson Marlow 620S 8

9 CellTank (CerCell) CellTank (CerCell) photo Probe Medium level in reservoir Red arrows = fluid circulation Circula6ng cell- free medium Rota6ng centrifugal pump Matrix where cells are cul6vated entrapped Source March 28, 2012 Véronique Chotteau, CETEG, KTH 9

10 CellTank 2202 system at CETEG March 28, 2012 Véronique Chotteau, CETEG, KTH 10

11 Experimental set-up TTF and ATF runs with Wave Bioreactor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`!$$ 16$$ ]Y$a$$$/,&;+,#$)N$I:+N%&0$;C"$:0%;:;%,&b$6 L $:--%;%,&$%&;,$C":-D*:/"$EL<58<<aG$ (0%;:;%,&$+:;"$\$+,/H%&0$:&0#"$$$ (K3$$$$L<5LR$+*'$\$R5[`$$ K33$$$$$L<5L[$+*'$\$R5X`$!.#;%I:;%,&$'"-%.'$$ :&%':#5/,'*,&"&;$M+""$cS$!26$!1$TP$'"-%.'!B%;C$CN-+,#ND:;"$Ec+I%&"$S/%"&;%M%/G$$ d$]$a$,m$cs5!26$3""-5!1$tp$ec+i%&"$s/%"&;%m%/g$ S.**#"'"&;:;%,&D$,M$0#./,D"$:&-$ ://,+-%&0$;,$/"##$/,&D.'*;%,&$ 0#.;:'%&"$$ (--%;%,&$,M$(&;%M,:'$!$ES(3!G$$.*$;,$Y<$**'$/,&/"&;+:;%,&$E),,D;$:--%;%,&$,+$)N$/,&;%&.,.D$*.'*%&0G$ (&:#ND"D$)N$_,I:$9%,*+,M%#"$3=@T$ /"##$-"&D%;Nb$I%:)%#%;Nb$/"##$-%:'";"+b$*2b$*!6Lb$,D',#:#%;Nb$/,&/"&;+:;%,&D$,M$0#./,D"b$ 0#.;:'%&"b$#:/;:;"$:&-$:'',&%:$ (&:#ND%D$,M$'()$/,&/"&;+:;%,&$ *+,;"%&$($2P=!$ Z$,+$*+"DD.+"$+%D%&0$M#,B$E(K3G$:&-$"^C:.D;$M#,B$E(K3G! March 28, 2012 Véronique Chotteau, CETEG, KTH 11

12 Experimental set-up CellTank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`8$'"-%.'!D%>E$EH-+,#H?=>"$7^+@%&"$_/%"&>%M%/;$$ a$j$]$,m$^_5!26$3""-5!1$`8$7^+@%&"$_/%"&>%m%/;$ _.**#"'"&>=>%,&?$,M$0#./,?"$=&-$ =//,+-%&0$>,$/"##$/,&?.'*>%,&$ 0#.>='%&"$$ (--%>%,&$,M$(&>%M,='$!$7_(3!;$$ T$ (&=#H?"?$)H$X,@=$<%,*+,M%#"$3Qb`$ /"##$-"&?%>Hc$@%=)%#%>Hc$/"##$-%='">"+c$*2c$*!6:c$,?',#=#%>Hc$/,&/"&>+=>%,&?$,M$0#./,?"c$ 0#.>='%&"c$#=/>=>"$=&-$='',&%=$ (&=#H?%?$,M$'()$/,&/"&>+=>%,&$ *+,>"%&$($28Q!$! March 28, 2012 Véronique Chotteau, CETEG, KTH 12

13 Results Perfusion using ATF or TFF in Wave Bioreactor TM March 28, 2012 Véronique Chotteau, CETEG, KTH 13

14 Continuation of run using TFF at very high cell density Viable cell density (MVC/mL) and viability (%) growth stabilized cell density growth stabilized cell density growth viability cell density me (days) 214 x 10 6 cells/ml TFF#10 - Viable cell density TFF#10 - Viability Cell density stabilized at 100 x 10 6 and 120 x 10 6 cells/ml by daily cell bleeds during > 2 weeks Cell densities 200 x 10 6 cells/ml (2 days) à Max cell density = 214 x 10 6 cells/ml Cell density limit due to limitations of membrane capacity for the encountered high viscosity (pressure = 1 bar), oxygenation and CO 2 level (31 kpa) March 28, 2012 Véronique Chotteau, CETEG, KTH 14

15 Perfusion using ATF or TFF at very high cell densities ATF : growth batch growth Viable cell density (MVC/mL) and viability (%) TFF : growth stabilized cell density growth stabilized cell density growth 132 x 10 6 cells/ml 214 x 10 6 cells/ml cell density (TFF) viability (ATF) viability (TFF) cell density (ATF) me (days) ATF#15 - Viable cell density TFF#10 - Viable cell density ATF#15 - Viability TFF#10 - Viability Max cell density = 132 x 10 6 cells/ml using ATF After maximum reached à cell density maintained at 100 x 10 6 cells/ml using ATF à Cell density limit due to pressure limitation to push highly viscous fluid using nonpressurisable disposable bioreactor March 28, 2012 Véronique Chotteau, CETEG, KTH 15

16 Total accumulated antibody production in the harvest, the cell bleed, the bioreactor and cell density using TFF Total produc7on (g) - Viable cell density (MVC/mL) ZOOM TOTAL accumulated production = harvest + loss in cell bleeds cell density 7me (days) TFF#10 - Viable cell density TFF#10 - TOTAL PRODUCTION = accumulated in harvest, bleeds & bioreactor TFF#10 - Accumulated produc6on in harvest with cell density at 25 x 1E6 cells/ml accumulated in harvest accumulated product loss in cell bleeds Observation of partial IgG retention by the hollow fiber filter Calculation exercise in this study run 17 days at x 10 6 cells/ml à 7 g 47 days at 25 x 10 6 cells/ml and 110 x 10 6 cells/ml à 96 g March 28, 2012 Véronique Chotteau, CETEG, KTH 16

17 Total accumulated antibody production in the harvest, the cell bleed, the bioreactor and cell density using TFF or ATF after 17 days at 25 x 10 6 cells/ml Total produc7on (g) TFF#10 - TOTAL PRODUCTION = accumulated in harvest, bleeds & bioreactor ATF#9 - TOTAL PRODUCTION = Accumulated produc6on in harvest, in bleeds and in bioreactor ATF#9 - Accumulated produc6on in harvest with cell density at 25 x 1E6 cells/ml TFF#10 - Accumulated produc6on in harvest with cell density at 25 x 1E6 cells/ml 4 TFF#10 - Accumulated loss in bleeds me (days) ATF#9 - Accumulated loss in bleeds Partial retention of IgG by hollow fiber filter yes yes Cell specific productivity (pg/cell/day) March 28, 2012 Véronique Chotteau, CETEG, KTH 17 ATF Total accumulated production Accumulated production in harvest (g) 9 7 Total removal of mab in cell bleeds/total production (w/w in %) Yield (production in harvest/total production) (w/w in %) Residual mab mass in bioreactor/total production (w/w in %) 6 15 TFF

18 Results Perfusion using CellTank TM March 28, 2012 Véronique Chotteau, CETEG, KTH 18

19 Cell density and perfusion rate in CellTank runs (BOL#1, BOL#2) %!!" $)!" $(!" $'!" $%!" $!!" )!" (!" '!" %!"!" Temperature #2 Cell density #2!"!" %" '" (" )" $!" $%" $'" $(" $)" %!" %%" %'" %(" %)" Trouble shooting!2#")*8&91-) Cell density #1 200 MVC/mL Perfusion rate #1 Perfusion rate #2 '!" &#" &!" %#" %!" $#" $!" #"!"#$"%&'(%")*+,-).)/"%0(1234)%&'")*5678&9-) BOL#1: Fast growth after day 14 (after trouble shooting (1 st run)) à up to 200 x 10 6 /ml BOL#2: Cell density kept 130 x 10 6 /ml at perfusion rate of 8-10 RV/day for over 10 days Temperature lowered from 37 C to 32 C/31 C/30 C/29 C on day 16 à cell growth arrest March 28, 2012 Véronique Chotteau, CETEG, KTH 19

20 IgG production in CellTank TM runs '#" )!!" '!" (!!"!"#$%&'%(')*+,&'$-."/01$ &#" &!" %#" %!" $#" $!" #"!"!" %" '" (" )" $!"$%"$'"$("$)"%!"%%"%'"%("%)"&!" 23.($-4+561$ *+,-$"."/012345" ":*;<=" *+,-$"."/012345" AB67=" *+,-%"."/012345" ":*;<=" *+,-%"."/012345" AB67="!""#$#%&'()*+,-*./0)#"102*3$,4* '!!" &!!" %!!" $!!" #!!"!"!" $" &" (" *" #!"#$"#&"#("#*"$!"$$"$&"$("$*"%!" 56$(*3)&784* +,-.#"/" " 9:;8216"<6;6" +,-.$"/" " 9:;8216"<6;6" Product accumulated with time and increasing cell density (after day 14 for BOL#1) Cell specific productivity in perfusion mode comparable to shake flask productivity except at 30 C where it was 40 % higher No retention of IgG in the polymer matrix.!"##$%&"'()'$&*+,-'./(01$2&34'"##4,516$ '"!# &"$# &"!# %"$# %"!#!"$# +,-.%#/#0122# # 489:;5<=6>?# +,-.&#/#0122# # 489:;5<=6>?#!"!#!# &# (# )# *# %!#%&#%(#%)#%*#&!#&&#&(#&)#&*#'!# March 28, 2012 Véronique Chotteau, CETEG, KTH 7(8"$2,51%6$ 20

21 Results Fed-batch versus perfusion using ATF or TFF March 28, 2012 Véronique Chotteau, CETEG, KTH 21

22 Comparison with fed-batch process Experimental set-up Initial à final volume = 2 L à 4 L Same set-points of DO, ph Temperature à 37 C (run FB#11) and 35.5 C from day 7 (run FB#16) Base medium = IS CHO CD XP medium with hydrolysate (Irvine Scientific) Feed medium = base medium + feed concentrate Efficient Feed A & B (InVitrogen) Viable cell density (MVC/mL) cell density 37 C cell density 35.5 C viability 37 C viability 35:5 C Viability (%) me (days) FB#11 - Viable cell density FB#16 - Viable cell density FB#11 - Viability FB#16 - Viability March 28, 2012 Véronique Chotteau, CETEG, KTH 22

23 Production in perfusion or fed-batch 4 L WAVE Bioreactor TM!"#$%&'(")*+,"-&./0& (%" ($" (#" (!" #&" #%" #$" ##" #!" '&" '%" '$" '#" '!" &" %" $" #"!" L9<N4C@=?"O :P"" -966":9?C@8E"'! & "3966CH5/"!" #" $" %" &" '!" '#" '$" '%" '&",12&.)$340& L9<N4C@=?"" -966":9?C@8E"" "#J"G"'! % "3966CH5/"! "#$%&'()*! *9:,M783A" )**+'!","-./-0/.)12" :";<=:43>=?"@?"A7<B9C8"D@8A"3966" :9?C@8E"78"F"'!!"G"'1%"3966CH5/".)*+I"," :";<=:43>=?"@?"A7<B9C8"D@8A"3966":9?C@8E"78"F" #J"G"'1%"3966CH5/" )**+'!"," :";<=:43>=?"@?"A7<B9C8"D@8A"3966":9?C@8E"78"F" #J"G"'1%"3966CH5/" *K+''","L<=:438"@?"M@=<9738=<" *K+'%","L<=:438"@?"M@=<9738=<" +#,-./012!345"!1,! 5""6! 70(*!)#88!$#2/0(9!'(!! +#,-./012!3)'8).8'(#$!-,1B! '2'89/#/!02!,.2!5""D>?6! 70(*!)#88!$#2/0(9!'(!!!!!!!!!!!!!!! B4&!E,1$.)(012! '-(#,!>F!$'9/!,.2! :!;!G! :!F!(1!H!G!!:!IJ!G! 3KBL!:!;;!GA7##M!E,1$.)#$!'(! >? N!)#88/ABC6! March 28, 2012 Véronique Chotteau, CETEG, KTH 23!

24 Results Cryopreservation from very high cell density perfusion March 28, 2012 Véronique Chotteau, CETEG, KTH 24

25 Cryopreservation study: set-ups Freezing conditions Growing cells at 100 x 10 6 cells/ml (from ATF) Cryo vial at 50 x 10 6 cells/ml Cryo vial at 100 x 10 6 cells/ml 45 % cell broth 45 % fresh medium 10 % DMSO 90 % cell broth 10 % DMSO After cryopreservation (N2 tank) à Cell thaw in shake flasks at 10 6 cells/ml à Study of cell revival and mab production Rm: freezing single experiment March 28, 2012 Véronique Chotteau, CETEG, KTH 25

26 Cell thaw after cryopreservation from high cell density culture ,5 90 Viable cell density (MVC/mL) 4 3,5 3 2,5 2 1,5 1 0, Viability (%) Cryo 1E8 c/ml - Cell density 7me (days) Cryo 0.5E8 c/ml - Cell density Cryo 1E8 c/ml - Viability Cryo 0.5E8 c/ml - Viability Cryopreservation from 100 x 10 6 cells/ml in vials of 100 or 50 x 10 6 cells/ml à excellent cell resuscitation March 28, 2012 Véronique Chotteau, CETEG, KTH 26

27 Cryopreservation: Production test in shake flasks 2 weeks after thaw IgG concentra6ons Viable cell density (MVC/mL) Cell densi6es mab (mg/l) Time (days) Cryo 1E8 c/ml - Cell density Cryo 0.5E8 c/ml - Cell density Cryo 1E8 c/ml - mab conc. Cryo 0.5E8 c/ml - mab conc. Normal mab production 2 weeks after thaw March 28, 2012 Véronique Chotteau, CETEG, KTH 27

28 Cells at very high cell density March 28, 2012 Véronique Chotteau, CETEG, KTH 28

29 Cell diameter at very high cell density 1. TTF perfusion run in Wave Bioreactor TM Viable cell density (MVC/mL) TFF#10 - ACD 100 TFF#10 - Viable cell density Time (days) Smaller cell diameter when cell density > 131 x 10 6 cells/ml 18,5 18,0 17,5 17,0 16,5 16,0 15,5 15,0 14,5 14,0 13,5 13,0 Average Cell Diameter (μ m) 2. Perfusion run in CellTank TM à cell diameter smaller when cell density 144 x 10 6 cells/ml (Fogale cell density probe - multi-frequency signal) March 28, 2012 Véronique Chotteau, CETEG, KTH 29

30 Distance between cells for different cell diameters!"#$%#"&'()(*+&",$')-'&.'',)$'##+)/%01) Calculation with hypothesis that cells are perfect incompressible spheres '!" $!" #+" &!" #*" #)" %!" #(" #'" $!" #&" #%" #!" #$" ##"!" #!" +" *" )" (" '" 11 &" 17 %" $" #" 20!"!,!-.!!" #,!-.!*" $,!-.!*" %,!-.!*"!'##)(',+*&2)/$'##+3041)!"#$%#"&'()(*+&",$')-'&.'',)$'##+)/%01) March 28, 2012 Véronique Chotteau, CETEG, KTH 30 #!!" +!" *!" )!" (!" /012"#!"32" /012"##"32" /012"#$"32" /012"#%"32" /012"#&"32"!,!-.!!" #,!-.!*" $,!-.!*" %,!-.!*" zoom!'##)(',+*&2)/$'##+3041) /012"#'"32" /012"#("32" /012"#)"32" /012"#*"32" /012"#+"32" /012"$!"32" 18 è when distance between cells becomes too small i.e. 2 µm à cells shrink è 250 x 10 6 cells/ml = limit for 16 µm diameter cells

31 Pressures during TTF perfusion Inlet pressure (before filter) Permeate pressure!"#$%&'($))*($&+,-(.& )% "#-% "#,% "#+% "#*% "#$% "#&% "#'% "#(% "#)% "%!"#)%!"#(%!"#'%!"#&%!"#$% Inlet pressure (before filter) Retentate pressure "% ("% &"% *"%,"% )""% )("% )&"% )*"% ),"% (""% (("% /0-,#$&1$##&2$")0%3&+456&1$##)789.& Permeate pressure./%0)%./%0(%./%0'% 1'%./0)% 1'%./0(% 1'%./0'% Retentate pressure March 28, 2012 Véronique Chotteau, CETEG, KTH 31 1)%./0)% 1)%./0(% 1)%./0'%

32 Viscosity very well correlated with cell density Viscosity = 0.01 ( Φ Φ 2 ) $&,-&.$&,-&.(& "#"+'%& Φ = volume fraction of cells in the mixture -./+&(01+##21+()341,( "#'& "#+& "#*&,-&.)& / & "#"%'%& "#"*'%& "#")'%& "#"('%&!"#$%#"&'()*%"#+,( Calculation for cell diameter 17 µm of viscosity of slurry according to Thomas D. G J. Colloid Science 20:267 è Increased viscosity due to increased cell density "#(& "#"$'%& "#""'%& è Increased pressure due to increased viscosity in a constricted filter section "& "&!"#""$%& ("& *"& +"& '"& $""& $("& $*"& $+"& $'"& (""& (("&!"43/+($+//(5+.#"&'()678($+//#9:;,( è Calculation of filter fiber numbers (or filter section) can be done given target cell density, allowed inlet pressure March 28, 2012 Véronique Chotteau, CETEG, KTH 32

33 Conclusions March 28, 2012 Véronique Chotteau, CETEG, KTH 33

34 Conclusions Cell density Very high cell density of 100 x 10 6 cells/ml stably maintained in growing phase and at high viability by cell bleeds in a perfused WAVE Bioreactor using TFF or ATF cell separations Very high cell density of 200 x 10 6 cells/ml achieved in CellTank Very high cell density of 130 x 10 6 cells/ml during 11 days with lower temperature in CellTank With present settings à maximal cell density = 214 x 10 6 cells/ml with TFF à maximal cell density = 132 x 10 6 cells/ml with ATF TFF à cell density limited by membrane capacity (for the encountered high viscosity), oxygenation and CO 2 level ATF à cell density limited by insufficient pressure to push highly viscous fluid when using nonpressurisable disposable bioreactor è TFF and CellTank allow reaching higher cell densities than ATF with present settings First time, CHO cell density 200 x 10 6 cells/ml in a wave-agitated bioreactor First time, CHO cell density 200 x 10 6 cells/ml in CellTank March 28, 2012 Véronique Chotteau, CETEG, KTH 34

35 Conclusions Cell density limit? Upper limit of cell density for suspension depends of cell diameter calculated theoretically for perfect spheres for CHO cells (diameter 16 µm) à 250 x 10 6 cells/ml smaller limit than tissue cells or adherent cells in absence of contact inhibition Applicable limit of cell density for suspension depends of cell diameter depends of equipment impact of cell shrinking? perhaps recommended to avoid shrinking limit of 130 x 10 6 cells/ml for CHO cells theoretical smallest distance between cells with unchanged diameter = 2 µm March 28, 2012 Véronique Chotteau, CETEG, KTH 35

36 Conclusions mab production No retention of mab in CellTank matrix using cell line #2 Retention using cell line #1 in hollow fibre filter: Higher retentions of mab by hollow fibre filter using TFF than ATF using In perfusion, major effect of this retention = loss of mab in the cell bleeds è CellTank the most favourable for production according to this study è ATF more favourable for production at stable cell density maintained by cell bleeds Potential production per bioreactor volume by perfusion much larger than fed-batch March 28, 2012 Véronique Chotteau, CETEG, KTH 36

37 Conclusions Operation Recommended to apply cell specific perfusion rate No cell sample today in CellTank (under development) Short residence time sec for TFF and ATF Short residence time 6-9 sec for CellTank Shear rate of 3400 s -1 well tolerated for TFF and ATF TFF ReadyToProcess disposable, easy to put in place and easy to put a new hollow fiber filter during cultivation à easier operation than ATF (autoclavable) Operation using CellTank easy and handy with robust integrated perfusion device Operation during 24 and 27 days could have been continued longer Absence of sparging in Wave Bioreactor TM and in CellTank in small scale à ADVANTAGEOUS The use of a single-use bioreactor equipped with robust cell separation device offers a solution alleviating technical and sterility challenges occurring in perfusion processes March 28, 2012 Véronique Chotteau, CETEG, KTH 37

38 Conclusions Hollow fiber operation Present study give mathematical tools to select / design hollow fibers lumen size and / or number of fibers à important for high cell density recommendation of larger lumen or larger number of fibers recommended to use larger number of fibers than 50 fibers (present study) using ATF for cell density x 10 6 cells/ml in disposable bioreactor filter area à impact on fouling March 28, 2012 Véronique Chotteau, CETEG, KTH 38

39 Perspectives - Applications High or very high cell densities of CHO cells, i.e. 50 to 130 x 10 6 cells/ml, are applicable for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arch 28, 2012 Véronique Chotteau, CETEG, KTH 39

40 Acknowledgements Sweden/USA Co-authors Marie-Francoise Clincke Carin Mölleryd Puneeth Samani Ye Zhang Co-authors Eva Lindskog Kieron Walsh Eric Fäldt Co-authors Per Stobbe Co-authors Christian Orrego Thanks to Atefeh Shokri Jan Kinnander Alexis Braun Thanks to Christian Kaisermayer Craig Robinson Swedish Governmental Agency for Innovation Systems March 28, 2012 Véronique Chotteau, CETEG, KTH 40