capto-adhere纯化工艺DOE实验设计PPT课件

1、CaptoTM adhere,Anovel multimodal anion exchange medium forlarge scale purification of monoclonal antibodies,Outline,Monoclonal antibody titer development CaptoTM adhere designed for MAb purification Screening/optimization of loading conditions Applications Different selectivity compared to tradition

2、al ion exchangers Selective removal of aggregates Two step purification of IgG1 from CHO cell cultute supernatant Viral clearance study Process economy Summary,Monoclonal antibody titer development,Fed-batch titer of 2,7g/L reported already in 1997 (Zhou et al) using GS gene expression system 1 to 2

3、g/L considered to be current industrial standard 10g/L estimated to be achieved in next 5 years,Mid 80s 0.05- 0.1 g/L,Mid 90s 0.5 -1 g/L,Currently 2-6 g/L,Future 10 g/L ,Higher titer implications on downstream purification,Higher cell densities More cell debris More contaminants host cell proteins D

4、NA viruses dimers/aggregates Greater quantity of MAb per batch,J.Bonnerjea, IBC Dublin Oct 23 2006,Removal of contaminants,High titers,High contaminant levels,Need for strong post Protein A polishing medium,CaptoTM adhere,Outline,Monoclonal antibody titer development CaptoTM adhere designed for MAb

5、purification Screening/optimization of loading conditions Applications Different selectivity compared to traditional ion exchangers Selective removal of aggregates Two step purification of IgG1 from CHO cell cultute supernatant Viral clearance study Process economy Summary,CaptoTM adhere,The design

6、of CaptoTM adhere,Is a strong multimodal 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 eco

7、nomy High load in flow-through mode (100 - 300 mg MAb/ml) Contaminant removal to formulation level in two chromatographic steps,CaptoTM adhere,Other 2-step processes,2-step processes suggested by: Wyeth Lonza (including Celltech) Tanox,2-step processes all contain the following chromatography steps:

8、 Protein A AIEX,Engineered Protein A,Protein A capture with MabSelect SuReTM,Highly productive two step process, The toolbox concept,WO 2004/076485 (Lonza,Outline,Monoclonal antibody titer development CaptoTM adhere designed for MAb purification Screening/optimization of loading conditions Applicati

9、ons Different selectivity compared to traditional ion exchangers Selective removal of aggregates Two step purification of IgG1 from CHO cell cultute supernatant Viral clearance study Process economy Summary,Flowthrough mode on CaptoTM adhere,mAU,Non-binding Partial binding,Antibody yield & Contamina

10、nt clearance,YIELD PURITY,OPTIMIZATION,Design of Experiments for optimization,Factors (inputs) Load (x1), pH (x2), Conductivity (x3,Responses (outputs) Yield (Y1), Dimer/aggregates (Y2) HCP (Y3), Protein A (Y4,Yield,D/A,Protein A,HCP,http:/,Optimization of loading conditions Design of Experiments,FA

11、CTOR SETTING Load Set according to goal: 75-300 mg/ml Conductivity ”Normal” range chosen: 2-15 mS/cm pH Initial experiments are required,GOAL With a load of 100 mg/ml Yield 90% Dimer/Aggregates 1% Protein A 5 ppm HCP 50 ppm,Optimization of loading conditions Lower pH limit in the design,pH 5.95,Samp

12、le loaded at pH 7.8 Load 1 mg/ml Elution in pH gradient from 7.8 to 4.0,lower pH in DoE: 6.0,pH 6.0, 2 mS/cm (green) Lower pH limit in DoE, 6.0 pH 8.0, 2 mS/cm (blue) Upper pH limit in DoE, 8.0,Sample load 75 mg/ml,Optimization of loading conditions Upper pH limit in the design,Optimization of loadi

13、ng conditions The Design,Linear and interaction models,Optimization of loading conditions The Design,Quadratic models,Yield,Load,pH,Cond,Load*pH,Load*Cond,Goal: High Yield in flowthrough pool,pH,Cond,Load,Dimer/aggregate clearance Goal 1,Load,pH*pH,pH,1,0,1,Goal: Low dimer/aggregate conc in flowthro

14、ugh pool,pH,Cond Not significant,Load,D/A conc in start material: 3.2,HCP clearance Goal 50ppm,Goal: Low HCP conc in flowthrough pool,pH,Cond,Load,HCP conc in start material: 206 ppm,Protein A clearance Goal 5 ppm,Goal: Low Protein A conc in flowthrough pool,pH,Cond,Load Not significant,Protein A co

15、nc in start material: 36 ppm,YIELD Conductivity 15 mS/cm,Dimer/Aggregates Conductivity 15 mS/cm,HCP Load 300 mg/ml,Protein A Load 300 mg/ml,Sweet spot analysis Gives the area (red) where the the goals are fulfilled. At a load of at least 100 mgMAb/ml resin: Yield in FT 90% D/A in FT 1% Protein A in

16、FT 5 ppm HCP in FT 50 ppm,DoE Summary,Outline,Monoclonal antibody titer development CaptoTM adhere designed for MAb purification Screening/optimization of loading conditions Applications Different selectivity compared to traditional ion exchangers Selective removal of aggregates Two step purificatio

17、n of IgG1 from CHO cell cultute supernatant Viral clearance study Process economy Summary,Different selectivity compared to traditional ion exchangers,CaptoTM adhere Elution pH: 6.2 - 4.9,Capto Q Elution pH: 9.9 6.2,Elution order: MAb4 MAb3 MAb 5 MAb 2 MAb 1,pH gradient elution of 5 MAbs,Clarified N

18、S0 cell culture supernatant 1.3 mg IgG1/ml pI 7.5 8.4 Capture on MabSelect Sure Elution pool D/A concentration 6,6% aggregates,Superdex 200 10/300,Selective removal of aggregates,Optimization of loading conditions,1) Binding mode, Elution by pH gradient,2) Flow-through mode + 2 pH units,pH 5.5,pH 7,

19、Load 100 200 mg/ml pH 5.5 7 Conductivity 10 50 mS/cm,Optimization by DoE,Effect of pH and conductivity on yield,Yield Controlled by pH Independent of Conductivity (10 50 mS/cm) Load (100 200 mg/ml,Effect of pH, conductivity and load on D/A-clearance,D/A clearance influenced by pH Higher pH Cond High

20、er cond Load Lower load Interaction pH cond,Conditions for D/A-clearance pH 6.5 and conductivity 30 mS/cm,High D/A clearance,Sample load 265 mg/ml D/A content was 6,Regeneration 0.1 M HAc pH 3.0,Selective removal of aggregates,SEC on Superdex 200 10/300,Flow through fractions Bound material,D/A cont

21、ent 60% D/A adsorbed to CaptoTM adhere,Dimers 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 preparations,D/A=dimers a

22、nd aggregates,Viral clearance study,IgG1 pool from MabSelect SuRe spiked with stock solutions of: MVM (Minute Virus 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,Virus clea

23、rance Capto adhere,Very good log10 reduction factors even for conditions where traditional ion exchangers do not work,Two step purification of IgG1,WO 2004/076485 (Lonza,Capture on MabSelect SuRe,Intermediate wash contained mainly D/A and HCP HCP reduced from 128300 to 55 ppm Protein A concentration

24、 1 ppm Aggregate content 0.7,Wash 25 mM NaP, 5% isopropanol,Anna Grnberg et al. “Screening and optimization of buffer conditions for Protein A chromatography using a 96-well format“ IBC conference in San Francisco (2006,Three step process,MabSelect SuRe Capto S Capto Q (bind /elute) (flow-through,Tw

25、o step process,MabSelect SuRe Capto adhere,MabSelect SuRe + Capto S + Capto Q * MabSelect SuRe + Capto adhere,Two vs three step purification,Capto adhere clearance,In most cases below limit of quantification, 1 ppb,Two step purification with 90-98% yieldProtein A and Capto adhere,Values within paren

26、thesis correspond to the initial levels (start values,MAb6 (CHO antibody feed): HCP was reduced from 9000 ppm to 2 ppm with 98 % yield,Outline,Monoclonal antibody titer development Capto adhere designed for MAb purification Screening/optimization of loading conditions Applications Different selectiv

27、ity compared to traditional ion exchangers Selective removal of aggregates Two step purification of IgG1 from CHO cell culture supernatant Viral clearance study Process economy Summary,Process economy study,The model process covers the unit procedures from harvesting to final formulation One 10,000

28、L fermentor Titer 5 g/L Resins and membranes list prices Labor set to $69/h Buffer cost, average, set to $2.0/L,Process economy, template,MabSelect SuRe,Capto adhere,SuperPro Designer Intelligen, Inc,Harvest/clarification,Virus inactivation,Virus filtration,UF/DF,Sterile filtration,Cell culture,Proc

29、ess economy and cost of production,Combination effect, use modern resins(capacity, flow, and lifetime improvements,Cost reduction 50% Process time down to 2-3 days,Working volume 10.000 L Titer 5g/L,0,5,10,15,20,25,30,MabSelect + SP and Q Sepharose Fast Flow Reference process 1,MabSelect SuRe + Capt

30、o S + Capto Q Reference process 2,MabSelect SuRe + Capto Q + Capto adhere 3-step process,MabSelect SuRe + Capto adhere 2-step process,Production cost contribution USD/kg,0,10,20,30,40,50,60,70,DSP process time h,Time,Outline,Monoclonal antibody titer development Capto adhere designed for MAb purific

31、ation Screening/optimization of loading conditions Applications Different selectivity compared to traditional ion exchangers Selective removal of aggregates Two step purification of IgG1 from CHO cell cultute supernatant Viral clearance study Process economy Summary,Summary,Capto adhere improves process economy by: Contaminant removal to formulation levels in one post-Protein A step Wide operational wi