captoadhere純化工藝DOE實(shí)驗(yàn)設(shè)計(jì)PPT課件

1、captotm adhereanovel multimodal anion exchange medium forlarge scale purification of monoclonal antibodies 2 /ge /2021-10-27outlinemonoclonal antibody titer developmentcaptotm adhere designed for mab purificationscreening/optimization of loading conditionsapplicationsdifferent selectivity compared t

2、o traditional ion exchangersselective removal of aggregatestwo step purification of igg1 from cho cell cultute supernatantviral clearance study process economysummary3 /ge /2021-10-27monoclonal antibody titer development fed-batch titer of 2,7g/l reported already in 1997 (zhou et al) using gs gene e

3、xpression system 1 to 2g/l considered to be current industrial standard 10g/l estimated to be achieved in next 5 years mid 80s 0.05- 0.1 g/lmid 90s 0.5 -1 g/l currently 2-6 g/lfuture 10 g/l ?4 /ge /2021-10-27higher titer implications on downstream purification higher cell densitiesmore cell debrismo

4、re contaminantshost cell proteinsdnavirusesdimers/aggregatesgreater quantity of mab per batchsupernatantmin log reductionhcp106 ng/mg 6 logsdna 106 ng/pg 6 logsmulv n/a20 logsmab aggregates 2-25% 1 logs j.bonnerjea, ibc dublin oct 23 20065 /ge /2021-10-27 removal of contaminantshigh titershigh conta

5、minant levelsneed for strong post protein a polishing mediumcaptotm adhere6 /ge /2021-10-27outlinemonoclonal antibody titer developmentcaptotm adhere designed for mab purificationscreening/optimization of loading conditionsapplicationsdifferent selectivity compared to traditional ion exchangersselec

6、tive removal of aggregatestwo step purification of igg1 from cho cell cultute supernatantviral clearance study process economysummary7 /ge /2021-10-27captotm adhere the design of captotm adhere+oon+ohohohoon+ohohohn-benzyl-n-methylethanolaminecaptotm high flow agarose8 /ge /2021-10-27 is a strong mu

7、ltimodal anion exchanger designed for: intermediate purification and polishing after capture on protein a removal of leached protein a dimers and aggregtes host cell proteins viruses nucleic acids . preferably in flowthrough mode. improves productivity and process economy high load in flow-through m

8、ode (100 - 300 mg mab/ml)contaminant removal to formulation level in two chromatographic stepscaptotm adhere9 /ge /2021-10-27other 2-step processes2-step processes suggested by:wyethlonza (including celltech)tanox2-step processes all contain the following chromatography steps:protein aaiex10 /ge /20

9、21-10-27engineered protein a1) time 0 2) 18 hour3) control1 2 3rprotein asureligandstability in cho cell lysate1) time 0 2) 18 hour3) control1 2 3rprotein asureligandstability in cho cell lysatestability cipmabselecttmmabselect suretm0.1m naoh 15 minstability cipmabselecttmmabselect suretm0.1m naoh

10、15 mineadcbprotein a capture with mabselect suretmghose et al., (2005) biotech bioeng 92, 665-673generic elution conditionsgeneric elution conditions11 /ge /2021-10-27 highly productive two step processmabselect sure pool for final filtration uf/dfcapto adherevirus inactivation & filtrationcell

11、removalcell culture12 /ge /2021-10-27mabselect sure aiex capto qciex capto spool for final filtration uf/dfcapto adherecapto adhereaiex capto q*virus inactivation & filtrationcell removalcell culture& the toolbox concept *wo 2004/076485 (lonza)13 /ge /2021-10-27outlinemonoclonal antibody tit

12、er developmentcaptotm adhere designed for mab purificationscreening/optimization of loading conditionsapplicationsdifferent selectivity compared to traditional ion exchangersselective removal of aggregatestwo step purification of igg1 from cho cell cultute supernatantviral clearance study process ec

13、onomysummary14 /ge /2021-10-27flowthrough mode on captotm adherestartwash startmaunon-binding partial bindingantibody yield&contaminant clearanceyield purityoptimization15 /ge /2021-10-27design of experiments for optimizationfactors (inputs)load (x1), ph (x2), conductivity (x3)responses (outputs

14、)yield (y1), dimer/aggregates (y2)hcp (y3), protein a (y4)yieldd/aprotein ahcpcond.loadphcond.loadphloadphhttp:/16 /ge /2021-10-27optimization of loading conditions design of experimentsfactor settingloadset according to goal: 75-300 mg/mlconductivity”normal” range chosen: 2-15 ms/cmphinitial experi

15、ments are requiredgoalwith a load of 100 mg/ml yield 90% dimer/aggregates 1% protein a 5 ppm hcp 50 ppm17 /ge /2021-10-27optimization of loading conditions lower ph limit in the designph 5.95 - sample loaded at ph 7.8- load 1 mg/ml- elution in ph gradient from 7.8 to 4.0lower ph in doe: 6.018 /ge /2

16、021-10-27ph 6.0, 2 ms/cm (green) lower ph limit in doe, 6.0ph 8.0, 2 ms/cm (blue) upper ph limit in doe, 8.0sample load 75 mg/mloptimization of loading conditions upper ph limit in the design19 /ge /2021-10-27loadphcond.6875300215optimization of loading conditions the design27187. 58. 58187. 58. 571

17、87. 5157187. 58. 56187. 58. 573008. 57758. 57187. 58. 57187. 51583001563001587515675283002630028752675condcond( ( m sm s/cm )/cm )phphloadload( m g/m l )( m g/m l )27187. 58. 58187. 58. 57187. 5157187. 58. 56187. 58. 573008. 57758. 57187. 58. 57187. 51583001563001587515675283002630028752675condcond(

18、 ( m sm s/cm )/cm )phphloadload( m g/m l )( m g/m l )linear and interaction models20 /ge /2021-10-27loadphcond.6875300215optimization of loading conditions the design27187. 58. 58187. 58. 57187. 5157187. 58. 56187. 58. 573008. 57758. 57187. 58. 57187. 51583001563001587515675283002630028752675condcon

19、d( ( m sm s/cm )/cm )phphloadload( m g/m l )( m g/m l )27187. 58. 58187. 58. 57187. 5157187. 58. 56187. 58. 573008. 57758. 57187. 58. 57187. 51583001563001587515675283002630028752675condcond( ( m sm s/cm )/cm )phphloadload( m g/m l )( m g/m l )quadratic models21 /ge /2021-10-276543210-1-2-3-4-5-6-7-

20、8%conductivity 2 ms/cmconductivity 8.5 ms/cmconductivity 15 ms/cmyield loadphcondload*phload*condgoal: high yield in flowthrough poolphcondload 22 /ge /2021-10-27dimer/aggregate clearance goal 1%loadph*phph-101%goal:low dimer/aggregate conc in flowthrough poolphcond not significant loadd/a conc in s

21、tart material: 3.2%23 /ge /2021-10-27hcp clearance goal 50ppm5-543210-1-2-3-4ppmloadcondphload 75 mg/mlload 187.5 mg/mlload 300 mg/mlgoal: low hcp conc in flowthrough poolphcond loadhcp conc in start material: 206 ppm24 /ge /2021-10-27protein a clearance goal 5 ppm-543210-1-2-3-4ppmph5condph*condgoa

22、l:low protein a conc in flowthrough poolphcond load not significant protein a conc in start material: 36 ppmyieldconductivity 15 ms/cmdimer/aggregatesconductivity 15 ms/cmhcpload 300 mg/mlprotein aload 300 mg/mlsweet spot analysisgives the area (red) where the the goals are fulfilled.at a load of at

23、 least 100 mgmab/ml resin:yield in ft 90%d/a in ft 1%protein a in ft 5 ppmhcp in ft 50 ppmdoe summary26 /ge /2021-10-27outlinemonoclonal antibody titer developmentcaptotm adhere designed for mab purificationscreening/optimization of loading conditionsapplicationsdifferent selectivity compared to tra

24、ditional ion exchangersselective removal of aggregatestwo step purification of igg1 from cho cell cultute supernatantviral clearance study process economysummary27 /ge /2021-10-27different selectivity compared to traditional ion exchangers captotm adhereelution ph: 6.2 - 4.9 6.07.08.09.010.011.00.01

25、0.020.030.040.050.060.0mlmab 1, ph 6.24mab 4, ph 9.86mab 3, ph 9.58mab 5, ph 9.41mab 2, ph 8.374.05.06.07.08.010.020.030.040.0mlmab 3: 5.25mab 1: 4.93mab 2: 5.12mab 5: 5.14mab 4: 6.22capto qelution ph: 9.9 6.2elution order: mab4 mab3 mab 5 mab 2 mab 1ph gradient elution of 5 mabs28 /ge /2021-10-27 0

26、 200 400 600 8001000mau0.05.010.015.0ml clarified ns0 cell culture supernatant 1.3 mg igg1/ml pi 7.5 8.4 capture on mabselect sure elution pool d/a concentration 6%6% aggregatessuperdex 200 10/300selective removal of aggregates29 /ge /2021-10-27optimization of loading conditions1) binding mode, elut

27、ion by ph gradient2) flow-through mode + 2 ph unitsph 5.5ph 7load 100 200 mg/ml ph 5.5 7conductivity 10 50 ms/cmoptimization by doe050100150200250mau4.06.08.010.012.0102030ml050100150200250mau4.06.08.010.012.0102030ml5.130 /ge /2021-10-27effect of ph and conductivity on yield yield controlled by ph

28、independent of conductivity (10 50 ms/cm) load (100 200 mg/ml)31 /ge /2021-10-27effect of ph, conductivity and load on d/a-clearance d/a clearance influenced by ph higher ph cond higher cond load lower load interaction ph cond conditions for d/a-clearance ph 6.5 and conductivity 30 ms/cm high d/a cl

29、earance32 /ge /2021-10-27sample load 265 mg/mld/a content was 6% regeneration 0.1 m hac ph 3.0 0 50010001500200025003000mau0.05.010.015.020.025.030.035.0mlinjectwashelutioncipselective removal of aggregates33 /ge /2021-10-27sec on superdex 200 10/300 flow through fractions bound material d/a content

30、 60% d/a adsorbed to captotm adhere ! 34 /ge /2021-10-27dimers and aggregates clearance sample load 120 mg/ml d/a reduced from 6 to 0.6% (10-fold) accumulated d/a 1% at sample load up to 200 mg/ml total yield of monomer 94% capto adhere has a high potential to selectively remove d/a from mab prepara

31、tions012345678060120150180265load mg igg/mlaccum d/a (%)load d/amg igg/ml%accumulated %start material6,0 600,70,681200,60,651500,90,721801,20,802652,21,30*d/a=dimers and aggregates*35 /ge /2021-10-27 viral clearance study igg1 pool from mabselect sure spiked with stock solutions of:mvm (minute virus

32、 of mice) single stranded dna virus non-enveloped, 20-26 nm mulv (murine leukemia virus) singel stranded rna enveloped, 80-110 nm applied to capto adhere in flow through mode 36 /ge /2021-10-27 virus clearance capto adhereviruscondlrf (ms/cm) 95% confidence iimitmvm105.8 0.3mvm305.9 0.3mulv104.5 0.4

33、mulv303.6 0.4very good log10 reduction factors even for conditions where traditional ion exchangers do not work!37 /ge /2021-10-27 two step purification of igg1mabselect sure aiex, capto qciex, capto spool for final filtration uf/dfcapto adhereaiex, capto q*capto adherevirus inactivation & filtr

34、ationcell removalcell culture*wo 2004/076485 (lonza)38 /ge /2021-10-27capture on mabselect sure01000200030004000a280 nm(mau)4.06.08.010.002004006008001000vol. (ml)ph01000200030004000a280 nm(mau)4.06.08.010.002004006008001000vol. (ml)ph intermediate wash contained mainly d/a and hcp hcp reduced from

35、128300 to 55 ppm protein a concentration 1 ppm aggregate content 0.7%.wash 25 mm nap, 5% isopropanolanna grnberg et al. “screening and optimization of buffer conditions for protein a chromatography using a 96-well format“ ibc conference in san francisco (2006) 39 /ge /2021-10-27three step process010

36、00200030004000a280 nm(mau)4.06.08.010.002004006008001000vol. (ml)ph01000200030004000a280 nm(mau)4.06.08.010.002004006008001000vol. (ml)ph 01000200030004000mau6.07.08.09.010.011.012.0 0 50100150200ml uv1_280nm cond ph inject logbook 0 5001000150020002500mau0.010.020.030.040.050.060.070.0mlwashelution

37、cip naohre-equilibrate 1re-equilibrate 2mabselect sure capto s capto q (bind /elute) (flow-through)40 /ge /2021-10-27two step processmabselect sure capto adhere41 /ge /2021-10-27both the two- and three step process successfully purified mab to impurity levels well acceptable for formulation*mabselec

38、t sure + capto s + capto q * mabselect sure + capto adheretwo vs three step purification42 /ge /2021-10-27 capto adhere clearanceprotein a eluatecapto adhere clearancehcp500 -10000 ppm2-3 logsprotein a5-100 ppm1-2 logs*dna 10-1000 ppb3-4 logs*mulvmvm n/a3.5-5.0 logs5.5-6.5 logsmab aggregates 1-10 %1

39、-2 logs*in most cases below limit of quantification, 1 ppb 43 /ge /2021-10-27two step purification with 90-98% yieldprotein a and capto adheremabcelllinepiloading conditionshcp50 ppmpra5 ppmd/a1%yieldphcondload1cho978187.512(206)0(36)0.5(3.3)902cho8.3-8.95.531502(10)2(260)0.6(2.1)953ns07.5-8.4621509

40、(85)0(0)0.8(1.5)954sp2/07.7-8.072010030(500-2000)0(1)0.15(0.14)915cho8.87.5202007.5(38)0(1) 50%process time down to 2-3 daysworking volume 10.000 ltiter 5g/l051015202530mabselect + sp and q sepharose fast flow reference process 1mabselect sure + capto s + capto qreference process 2mabselect sure + c

41、apto q + capto adhere3-step processmabselect sure + capto adhere 2-step process production cost contribution usd/kg010203040506070dsp process time htime49 /ge /2021-10-27outlinemonoclonal antibody titer developmentcapto adhere designed for mab purificationscreening/optimization of loading conditionsapplicationsdifferent selectivity compared to traditional ion exchangersselective removal of aggregatestwo step purification of igg1 from cho cell cultute supernatantvi