A novel multimodal anion exchanger designed for monoclonal antibody purification

Transcription

1 A novel multimodal anion exchanger designed for monoclonal antibody purification IBC s 18th International, Antibody Development &Production February-March, 2007 Hans J Johansson, Anders Ljunglöf, Kjell Eriksson, Eggert Brekkan, Gustav Rodrigo and Tomas Björkman GE Healthcare Bio-Sciences AB Uppsala, Sweden

2 Outline Capto TM adhere designed for mab purification Screening/optimization of running conditions Removal of: aggregates Host cell proteins DNA leached Protein A Virus Bind-elute mode Two step purification Summary 2/

3 The toolbox concept Cell culture Cell removal MabSelect SuRe Virus Inactivation & Filtration CIEX Capto S AIEX Capto Q Capto adhere AIEX Capto Q* Capto adhere Pool for Final Filtration UF/DF *WO 2004/ (Lonza) 3/

4 The design of Capto TM adhere A multimodal strong anion exchanger for Intermediate purification and polishing after capture on Protein A Capto TM High flow agarose + OH Electrostatic OH O O N + OH Hydrophobic Aromatic H-Bond Donor Acceptor N-Benzyl-N-methylethanolamine 4/

5 Design of Experiments for optimization Factors (inputs) Load (x 1 ), ph (x 2 ), Conductivity (x 3 ) ph Load Cond. Yield D/A Responses (outputs) Yield (Y 1 ), Dimer/aggregates (Y 2 ) HCP (Y 3 ), Protein A (Y 4 ) Protein A HCP 5/

6 Optimization of loading conditions Design of Experiments GOAL With a load of 100 mg/ml Yield > 90% Dimer/Aggregates 1% Protein A 5 ppm HCP 50 ppm FACTOR SETTING Load Set according to goal: mg/ml Conductivity Normal range chosen: 2-15 ms/cm ph Initial experiments are required 6/

7 Optimization of loading conditions Lower ph limit in the design ph 6.0 lower ph in DoE: 6.0 Sample loaded at ph 7.8 Load 1 mg/ml Elution in ph gradient from 7.8 to 4.0 7/

8 Optimization of loading conditions Upper ph limit in the design Sample load 75 mg/ml 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 8/

9 Optimization of loading conditions The Design 8 ph 6 75 Load Cond. Load (mg/ml) Linear and interaction models ph Cond (ms/cm) /

10 Outline Capto TM adhere designed for mab purification Screening/optimization of running conditions Removal of: aggregates Host cell proteins DNA leached Protein A Virus Bind-elute mode Summary 10 /

11 Selective removal of aggregates Clarified NS0 cell culture supernatant 1.3 mg IgG 1 /ml pi Capture on MabSelect Sure Elution pool D/A concentration 6% mau Superdex /300 6% aggregates ml 11 /

12 Selective removal of aggregates mau Conditions for D/A-clearance ph 6.5 and conductivity 30 ms/cm 3000 Sample load 265 mg/ml D/A content was 6% Regeneration 0.1 M HAc ph Inject Wash Elution CIP ml 12 /

13 D/A- clearance Results D/A Load D/A mg IgG/ml % Accumulated % Start material 6,0 60 0,7 0, ,6 0, ,9 0, ,2 0, ,2 1,30 Accum D/A (%) Load mg IgG/ml 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 13 /

14 Removal of CHOP & DNA Purification of an IgG1 under non-binding conditions, ph 6.5 and 4.5 ms/cm. 14 /

15 Analytical results from three different materials Load material Conditioning Sample Yield CHOP Aggreg. Protein A DNA (%) (ng/mg) (%) (ng/mg) (pg/mg) Protein A pool* UF/DF Load (100) < Pool <1.95 <1 Protein A pool* Direct cond. Load (100) Pool (90) <1.95 <1.1 CIEC pool** Direct cond. Load (100) <1.95 Pool 100 *load: 50 mg/ml resin **load: 100 mg/ml resin <4 0.5 <1.95

16 Removal of leached Protein A Start material: Protein A purified human IgG 1 Protein A: 1100 ppm Dimer content: 2.8 % pi of mab: Load: 150 g/l Sweet spot analysis: Protein A < 5 ppm yield > 92 % Dimer/aggregates < 1 % x 16 /

17 Example of chromatography run in the DoE A280 nm (mau) Loading condition: Flowthrough ph 6.7 Salt conc. 50 mm NaCl Load 150 mg Protein A: 2 ppm Vol.(ml) D/A: 0.5% Yield: 96% 17 /

18 Viral clearance study IgG 1 pool from MabSelect SuRe spiked with stock solutions of: MVM (Minute Virus of Mice) Single stranded DNA virus Non-enveloped, nm MuLV (Murine leukemia Virus) Singel stranded RNA Enveloped, nm Applied to Capto adhere in flow through mode 18 /

19 Virus clearance* Capto adhere Virus Cond LRF (ms/cm) 95% confidence Iimit MVM ± 0.3 MVM ± 0.3 MuLV ± 0.4 MuLV ± 0.4 Very good log reduction factors even for conditions where traditional ion exchangers do not work! *Study performed by NewLab BioQuality AG 19 /

20 Outline Capto TM adhere designed for mab purification Screening/optimization of running conditions using factorial design Removal of aggregates, host cell proteins, DNA, leached Protein A and viruses Bind-elute mode Two step purification Summary 20 /

21 Bind-elute mode compared to AIEC Capto TM adhere Elution ph: Capto Q Elution ph: Elution order: MAb 4 > MAb 3 > MAb 5 > MAb 2 > MAb Mab 2: 5.12 Mab 1: Mab 3, ph 9.58 Mab 5, ph Mab 4, ph Mab 3: 5.25 Mab 5: Mab 2, ph Mab 4: Mab 1, ph ml ml 21 /

22 Combined ph and conductivity shift for elution Load: 25 g/l Buffer A: 25 mm phosphate, ph 7.5 Buffer B: 50 mm acetate, 300 mm NaCl, ph 5.5 UV1_280nm Cond ph Fractions Inject Logbook After adhere Start Flowthrough mau mau Analysis by SEC: RED Start material Blue Product peak Green flow through Wash Elution Strip CIP NaOH Re-equilibrate 1 Re-equilibrate F2 Waste F3 F4 Waste ml ml 22 /

23 Elution by linear or step gradient Yield (%) D/A (%) PrA (ng/ml) PrA (ppm) HCP (ng/ml) Start Gradient elution FT Not analyzed Eluate <3 <3 Strip Not analyzed Step elution FT Not analyzed Eluate <4 <3 Strip Not analyzed 23 /

24 Outline Capto TM adhere designed for mab purification Screening/optimization of running conditions Removal of: aggregates Host cell proteins DNA leached Protein A Virus Bind-elute mode Two step purification Summary 24 /

25 Two steps purification process Cell culture Cell removal MabSelect SuRe Virus Inact.& Filtr. multi-modal AIEC Increased selectivity Wider window of operation Improved productivity Final Filtration UF/DF 25 /

26 Protein A capture with MabSelect SuRe Engineered Protein A Stability in CHO cell lysate E D A B C rprotein A SuRe ligand ) Time 0 2) 18 hour 3) Control Stability CIP Generic elution conditions MabSelect TM MabSelect SuRe TM 0.1M NaOH 15 min Ghose et al., (2005) Biotech Bioeng 92, /

27 Capture on MabSelect SuRe A280 nm (mau) 4000 Wash 25 mm NaP, 5% isopropanol ph Vol. (ml) Intermediate wash contained mainly D/A and HCP HCP reduced from to 55 ppm Protein A concentration < 1 ppm Aggregate content 0.7%. 27 /

28 Two step process MabSelect SuRe Capto adhere Two step process Accumulated D/A Protein A HCP yield (%) (%) (ppm) (ppm) Start material spiked with HCP MabSelect SuRe 95 <0.7 <1 55 Capto adhere 90 <0.1 n.q /

29 Two step purification with 90-98% yield Protein A and Capto adhere MAb Cell line pi Loading conditions ph Cond Load HCP <50 ppm PrA <5 ppm D/A <1% Yield 1 CHO ~ (206) 0 (36) 0.5 (3.3) 90 2 CHO (10) <2 (260) 0.6 (2.1) 95 3 NS (85) 0 (0) 0.8 (1.5) 95 4 SP2/ ( ) 0 (<1) 0.15 (0.14) 91 5 CHO (38) 0 (<1) <0.1 (0.7) Values within parenthesis correspond to the initial levels (start values) /

30 Process economy study Cell culture The model process covers the unit procedures from harvesting to final formulation One 10,000 L fermentor Titer 5 g/l Resins and membranes, cost according to list prices Labor set to $69/h Buffer cost, average, set to $2.0/L Cell removal MabSelect SuRe Virus Inact.& Filtr. Capto TM adhere UF/DF, Sterile filtration 30 /

31 Process economy, template Diluant Retentate S-122 SuperPro Designer Intelligen, Inc. S-108 S-101 S-102 S-107 P-1 / V-101 Cell Cell culture P-06 / DF-101 Clarification P-06b / DE-101 P-8 / V-110 Depth Filtration Blending / Storage Harvest/clarification S-127 P-2 / V-102 Blending / Storage S-112 Prot A Equilib Prot A Wash ProtA Elute Prot A Strip S-103 S-110 P-05 / C-101 Capture- MabSelect MabSelect SuRe PnA Waste S-120 Citric Acid P-4 / V-105 ph adjustment P-07 / V-103 NaOH Virus inactivation NaCl S-109 Virus inactivation S-114 P-19 / V-111 Blending/Storage S-123 S-126 S-124 S-125 P-15 / C-103 S-121 Removal & Polishing- Adhere Capto adhere S-134 S-106 P-9 / V-104 Blending/Storage S-115 S-131 S-105 P-6 / DE-102 Dead-End Filtration S-111 S-113 P-11 / V-107 Pool Virus filtration P-18 / DE-03 Virus Filtration S-109D S-136 P-3 / V-108 Storage S-135 P-5 / DF-103 Diafiltration UF/DF S-133 S-128 S-132 P-7 / DE-103 P-10 / V-109 Bioburden Reduction Sterile filtration Mobile Storage D S-129 S /

32 Process economy and cost of production Production cost contribution [USD/kg] Working volume L Titer 5g/L Cost reduction > 50% Process time down to 2-3 days Time DSP process time [h] 0 MabSelect + SP and Q Sepharose Fast Flow Reference process 1 MabSelect SuRe + Capto S + Capto Q Reference process 2 MabSelect SuRe + Capto Q + Capto adhere 3-step process MabSelect SuRe + Capto adhere 2-step process 0 32 /

33 Summary Capto adhere contaminant clearance Protein A eluate Capto adhere Clearance HCP ppm 2-3 logs Protein A ppm 1-3 logs DNA ppb 3-4 logs* MuLV MVM MAb aggregates n/a logs logs 1-10 % 1-2 logs *In most cases below limit of quantification, < 1 ppb 33 /

34 Summary Capto adhere a new tool in the box (not only for antibodies) Facilitates development of a two-step process Wide operational window of ph and conductivity Robust alternative to hydroxy apatite Cell culture Cell removal MabSelect SuRe Virus Inactivation & Filtration Capto adhere Pool for Final Filtration UF/DF 34 /

35 Acknowledgements Martin Antti, Laura Chirica, Liv Johansen, Bo-Lennart Johansson, Jean-Luc Maloisel, Elin Monie, Elisabeth Wallby Boehringer Ingelheim Pharma 35 /

36 GE Healthcare Bio-Sciences AB, a General Electric Company. GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden MabSelect, MabSelect SuRe,Capto, Superdex and Sepharose are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram are trademarks of General Electric Company. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. 36 /