Continuous Chromatography for Monoclonal Antibody Purification from Cell Culture Supernatant

Continuous Chromatography for Monoclonal Antibody Purification from Cell Culture Supernatant Massimo Morbidelli Institute for Chemical and Bioengineering, ETH Zurich, Switzerland www.morbidelli.ethz.ch

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. MAb purification from cccs using ion-exchange 5. MAB Variant Separation 6. Comparison of technologies 2

1. MAb purification challenge MAb provided by Merck-Serono: mab contains variants IEF: analyt. CIEX (Propac wcx): pi range of mab variants: 7.4-8.2 3

1. MAb purification challenge MAb obtained from Merck-Serono: analyt. CIEX (Propac wcx): Batch pools: Red: CIEX Blue: Protein A mab fragments, early eluting in CIEX 4

1. MAb purification challenge MAb obtained from Merck-Serono: analyt. SEC (Tosoh): Red: Protein A purif. mab Blue: clarified supernatant Aggregates (early eluting in SEC) 5

1. MAb purification challenge MAb obtained from Merck-Serono: SEC-Analysis of fractionation of preparative mab gradient elution: Aggregates are late eluting in CIEX. 12 mab conc [g/l] 0.3 conc mab, Mononer [g/l] 10 8 6 4 2 Monomer Dimer Trimer 0.25 0.2 0.15 0.1 0.05 conc Agg [g/l] 0 80 85 90 95 time [min] 0 6

1. MAb purification challenge Monoclonal Antibody purification from cell culture supernatant: Cell Culture supernatant is multi-component (fragments, Aggregates, HCP, DNA) Pure MAb is multi-component (variants) Purification of a mixture from a mixture Monoclonal Antibody (mab): 150 kda 7

1. MAb purification challenge Summary (CIEX): conc. [maus] (mab) 16000 14000 12000 10000 8000 6000 4000 2000 mab Propac A280 W2 W3 2000 1800 1600 1400 1200 1000 800 600 400 200 conc. [maus] ( fragments), [mau] (online) conc mab, Mononer [g/l] 12 10 8 6 4 2 mab conc [g/l] Monomer Dimer Trimer 0.3 0.25 0.2 0.15 0.1 0.05 conc Agg [g/l] 0 0 47 49 51 53 55 57 59 61 time [min] 0 80 85 90 95 time [min] 0 Product conc. Weak (Fragments + HCP) Strong (Aggregates) time Three-fraction separation required to purify product. 8

1. MAb purification challenge Batch chromatography: If the desired purity is high, the achieved yield will be low! Product conc. Weak (Fragments + HCP) Strong (Aggregates) Change the resin (e.g. use affinity chromatography) time Or Use a different process 9

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. Application examples 5. Comparison of technologies 10

2. Continuous Liquid Chromatography Batch versus continuous chromatography: - selective adsorption leads to different migration velocities fast component liquid flow chromatographic column Features: Linear gradients Three fraction separations slow component 11

2. Continuous Liquid Chromatography Batch versus continuous chromatography liquid flow slow solid flow 12

2. Continuous Liquid Chromatography Batch versus continuous chromatography liquid flow fast solid flow 13

2. Continuous Liquid Chromatography From batch to continuous countercurrent chromatography liquid flow? intermediate solid flow 14

2. Simulated Moving Bed Chromatography True Moving Bed Design the unit with respect to an observer moving with the solid 15

2. Simulated Moving Bed Chromatography SMB scheme: Raffinate (early eluting) 4 4 Eluent 1 3 1 3 Feed 2 2 Extract (strongly adsorbing) 16

2. Batch versus Continuous Chromatography Separation of a pharmaceutical intermediate racemate mixture on a chiral stationary phase (CSP) 1 3 2.5 2 1.5 1 0.5 HPLC Batch SMB -80% 8x 1 J.Chrom A 1006 (1-2): 267-280, 2003 0 Eluent need [L/g] Solvent requirement Productivity Productivity [g/ kg/min] 17

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. MAb purification from cccs using ion-exchange 5. Comparison of technologies 18

3. Evolution of technologies Batch chromatography: multi-fraction separation linear solvent gradients pulsed feed low efficiency SMB: continuous feed counter-current operation high efficiency binary separation step solvent gradients MCSGP (Multi-column Countercurrent Solvent Gradient Purification): 19

3. MCSGP - Principle Product strong weak conc. Elution time 20

3. Principle 6 Column Purification unit all H out all P out no H out all L out no P out no P out 21

3. Principle 6 Column Purification unit all H out all P out no H out all L out no P out no P out 22

all P out all L out no P out all H out no H out no P out 23

3. Semicontinuous 3-Column Operation all P out all L out no P out all H out no H out no P out 24

3. Mobile MCSGP Unit 3 column MCSGP Process - columns, multiposition valves, gradient pumps - UV/Cond./pH Monitor - control computer - based on Aekta/ Unicorn - worldwide patent pending 25

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. MAb purification from cccs using ion-exchange 5. MAb Variant Separation 6. Comparison of technologies 26

4. Results: MAb capture from cccs Comparison of batch Protein A chromatography and MCSGP (commercial HCP ELISA) 100.0% 95.0% MCSGP 90.0% Yield 85.0% 80.0% Increase purity Protein A 75.0% 70.0% 0 500 1000 1500 2000 2500 HCP pool [ppm] 27

4. Results: MAb capture from cccs Comparison of batch Protein A chromatography and MCSGP Conc. Purity Yield Prod. Pool Pool Pool Mode Resin type SN dil cmab HCP [x-fold] [g/l] [ppm] [%] norm. Batch Aff. Mab Select Sure PA 1 4.8 2036 82.0% 1* 1st step MCSGP Resin 1 run A SO3 4 2.7 146 94.9% 1.1 MCSGP Resin 2 run B SO3 4 4.7 226 96.1% 4.8 MCSGP Resin 2 run C SO3 3 4.9 625 96.0% 5.0 * Productivity of all runs normalized to Protein A run productivity MCSGP has ca. 10x higher HCP-clearance than Protein A MCSGP reduces HCP by 2-3 logs 28

4. Results: Aggregate clearance Excellent aggregate clearance Aggregate content: Protein A: 0.8% MCSGP: 0.4% Size exclusion chromatogram: Tosoh TSKgel G3000SWXL 29

4. Results: Second purification step Polishing CaptoAdhere (ph grad. 8.0-4.0) : conc [g/l], A280 calibrated 2.5 2.0 1.5 1.0 0.5 0.0 mab Eluate A280 imp cond ph Capto Adhere 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 cond [ms/cm], ph*10 [-] 0 20 40 60 80 100 time [min] Purity of Pool: 99.5%, Yield 93.4 % 30

4. Results: Full purification CIEX-MCSGP capture samples purified with Capto Adhere Conc. Purity Yield Prod. Pool Pool Pool Mode Resin type SN dil cmab HCP [x-fold] [g/l] [ppm] [%] norm. Batch Aff. Mab Select Sure PA 1 4.8 2036 82.0% 1* 1st step MCSGP Resin 1 run A SO3 4 2.7 146 94.9% 1.1 MCSGP Resin 2 run B SO3 4 4.7 226 96.1% 4.8 MCSGP Resin 2 run C SO3 3 4.9 625 96.0% 5.0 2nd step Batch Adhere polish A n.a. 3 2.0 1 96.1% 1.4 Batch Adhere polish B n.a. 3 2.4 2 95.8% 2.3 Batch Adhere polish C n.a. 3 2.2 3 94.3% 1.9 * Productivity of all runs normalized to Protein A run productivity Final product after 2-step process in specification (10 ppm) 31

4. Results: Full purification Polishing Capto Adhere (complete removal of fragments): Pink: cccs Blue: MCSGP Red: MCSGP+Adh. Green: Final Product Serono Analytical CIEX (Propac wcx-10, 4 x 250 mm) 32

4. Results: MAb capture from cccs Comparison of Pool fractions and purest fractions (Protein A analysis): Yield 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CIEX batch chromatography CIEX MCSGP Purest fraction in CIEX batch 95% 96% 97% 98% 99% 100% Purity 33

5. Summary - MCSGP 3-step process replaced by 2-step process Protein A CIEX BE AIEX FT MCSGP CIEX MMA BE 34

4. Results: MCSGP MCSGP: Internal recycling High yield and purity are achieved simultaneously. concentration vw W P S time Yield 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CIEX batch chromatography CIEX MCSGP Purest fraction in CIEX batch 95% 96% 97% 98% 99% 100% Purity 35

4. MAb capture from cccs (including CIP) 4 columns required (3 columns perform purification, 1 is CIPped) CIP purification Q CIP Q 2, c 2 Q 4, c 4 Q 6, c 6 CCL: 8 2 4 6 CIP Q Equil Q 1, c 1 Q 3, c 3 Q Feed D BL: 7 1 3 5 CIP S P W 36

4. MAb capture from cccs (including CIP) After 120 hrs of operation, headspace is visible in one column (total cumulated operating time of columns up to that point: 400 hrs) Pressure drop starts to increase 0.30 Pressure drop [MPa] 0.25 0.20 0.15 0.10 CIP no CIP 0.05 0.00 0 2000 4000 6000 8000 time [min] 37

4. MAb capture from cccs (including CIP) After 7200 min of operation, CIP with NaOH is re-established Headspace disappears Pressure drop reduced back to normal 0.30 Pressure drop [MPa] 0.25 0.20 0.15 0.10 0.05 CIP no CIP CIP 0.00 0 2000 4000 6000 8000 10000 time [min] 38

4. MAb capture from cccs (including CIP) CIP required for DNA clearance purity [ng/mg] 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 DNA/MAb [ng/mg] HCP/MAb [ng/mg] c Mab [g/l] c Mab (A280) [g/l] CIP no CIP CIP 0.160 0.140 0.120 0.100 0.080 0.060 0.040 0.020 concentration [g/l] 0 0 2000 4000 6000 8000 10000 0.000 time [min] 39

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. MAb purification from cccs using ion-exchange 5. Mab Variant Separation 6. Comparison of technologies 40

5. Antibody Variant Separation 3 MAB variants with a variation in the constant part of the molecule Characterization and design of preparative separation by ion-exchange K K K I 1 F 1 F 2 I 2 F 3 Analytical Chromatogram on Propac WCX-10, ph 6.3, A 220 41

5. Linear Gradient Elution Experiment Simulation Purity of F2 < 80 % Required: Purity F2 > 80 % Yield F2 > 90 % Continuous Process Fractogel EMD COO 100x4.6 mm, d p = 30µm 42

5. MCSGP Purity and Yield of MAB Variant Process Start-up Purity of F2 in Fraction P Purity of F2 [-] 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% purity F2 yield F2 300 0 5 10 15 20 25 time [h] 250 Feed A220 [mau]_ 200 150 100 50 F2 in P fraction Purity of F2 = 93% Yield of F2 = 93% 0 3 5 7 9 time [min] 43

5. Trends in mab production The ratio of the variants may be influenced by changing the switch time tcc Deamidated variants Protein A Pool contains all variants Product variants 44

5. Trends in mab production mab product purity defined by bacteria! Staphylococcus aureus 45

Content 1. MAb purification challenge 2. Introduction to continuous liquid chromatography 3. Continuous gradient chromatography (MCSGP) 4. MAb purification from cccs using ion-exchange 5. MAB Variant Separation 6. Comparison of Technologies 46

5. Comparison of technologies Productivity as a function of mab titer Productivity productivity [g/l/min] [g/l/h] 100 90 80 70 60 50 40 30 20 10 0 MCSGP no dilution MCSGP 2x dilution Protein A batch MCSGP 3x dilution 0 2 4 6 8 10 12 14 16 mab mab titer [g/l] Experimental Results: SN with 14 g/l mab, no dilution SN with 2.5 g/l, dil. 1:4 Results obtained from collaboration 47

5. Trends in mab production Affinity and CIEX resin costs ($ per g mab): $7.0 resin costs [US $ / g mab] $6.0 $5.0 $4.0 $3.0 $2.0 $1.0 Protein A batch, $ 20000 / L Protein A batch, $ 10000 / L MCSGP 3x dilution MCSGP 2x dilution MCSGP no dilution $0.0 0 2 4 6 8 10 12 14 16 mab titer [g/l] 48

6. Summary Continuous processes outperforms batch processes in terms of yield, purity and productivity All processes using Protein A can not relieve the cost pressure for increasing mab titers MCSGP with ion-exchange reaches purity and yield comparable to Protein A batch chromatography, and raises productivity MCSGP can affect the MAB Safety and Potency 49

6. Acknowledgements Industrial Partners: Merck Serono, Switzerland Novartis, Switzerland Lonza, UK Chromacon AG, Switzerland PhDs & Postdocs in preparative chromatography: Aumann, Dr. Lars Müller-Späth, Thomas Ströhlein, Dr. Guido 50