Multicolumn Continuous Countercurrent Chromatography

Multicolumn Continuous Countercurrent Chromatography
Massimo Morbidelli Institute for Chemical and Bioengineering, ETH Zurich, Switzerland Integrated Continuous Biomanufacturing 2013, 20th – 24th Oct, Barcelona

Countercurrent Chromatography for three stream purifications
Outline Process evolution: from batch to multicolumn simulated moving bed chromatography Countercurrent Chromatography for three stream purifications Countercurrent Chromatography for highly selective stationary phases Application examples Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Batch chromatography Pulsed feed of mixture Fixed stationary phase
fast component Pulsed feed of mixture Fixed stationary phase Chromatography is intrinsically discontinuous chromatographic column liquid flow slow component Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Continuous chromatography
Continuous feed of mixture Fixed stationary phase (multiple columns requires) liquid flow fast component slow component Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Countercurrent principle (periodic)
Pulsed feed of mixture (simulated) movement of stationary phase liquid flow (simulated) moving bed Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Countercurrent principle (continuous)
Continuous feed of mixture (simulated) movement of stationary phase liquid flow (simulated) moving bed Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Batch Chromatography Selective adsorption leads to different elution velocities: select switch times Features: Linear gradients Three fraction separations fast component chromatographic column liquid flow slow component Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Continuous Countercurrent Chromatography
Selective adsorption leads to different elution velocities: select solid speed ? liquid flow solid flow Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Simulated Moving Bed Chromatography
The SMB scheme: Eluent Raffinate (early eluting) 4 4 3 1 3 1 2 2 Feed Extract (strongly adsorbing) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Batch versus SMB performance
Separation of a pharmaceutical intermediate racemate mixture on a chiral stationary phase (CSP)1 8x -80% Eluent need [L/g] Productivity [g/ kg/min] 1 J.Chrom A 1006 (1-2): , 2003 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Countercurrent chromatography for ternary separations (polishing applications)

Typical bio-purification problem
Example: mAb purification from cell culture supernatant typical chromatogram for mAb elution on cation-exchanger: mAb HCPs aggregates fragments Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Purification challenge
Generic purification problem: separate into 3 fractions #2: mAb #3: late eluting impurities #1: early eluting impurities Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

in 3-fraction batch chromatography: intrinsic trade-off between yield and purity! high yield, low purity high purity, low yield Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

in 3-fraction batch chromatography: intrinsic trade-off between yield and purity! Alternatives: - Very Selective Stationary Phase (eg, Protein A) - Continuous Countercurrent Chromatography (MCSGP) process yield alternatives ? purity Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Combining batch and SMB
Batch chromatography: SMB:  multi-fraction separation  linear solvent gradients  continuous feed  counter-current operation  high efficiency  pulsed feed  low efficiency  binary separation  step solvent gradients MCSGP (Multi-column Countercurrent Solvent Gradient Purification): Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

The MCSGP principle: recycle until it‘s pure
Conventional single column batch chromatography MCSGP chromatography time more pure product Reprocess impure product time pure product impure product to waste |

6-column continuous MCSGP unit Gradient Batch Process
concentration gradient P (target protein) W S vW time re-equilibrate load Feed & wash start gradient recycle weak fraction collect pure target protein cleaning in place (CIP) pre-load with weak fraction elute & recycle strong fraction Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Principle 6 Column Purification unit
Load // elute light elute overlapping product/light elute product elute overlapping heavy/product elute heavy Receive overlapping product/light 5 4 3 2 1 6 L P H c inerts t t t t tF Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Animation 6 Column MCSGP unit
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

4-column process (separate CIP position)
Counter-current (CCL) and batch (BL) are alternating, but additional CIP position for column CIP purification Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Animation 3-Column MCSGP simplified

The MCSGP principle with (only) two columns
    Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Contichrom® & MCSGP explained
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 24

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 25

Feed waste waste product & waste product & waste product Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 26

Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: W-impurities Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 27

Eluent Inline Dil. Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 28

Eluent Feed Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Product Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 29

Eluent Inline Dil. Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 30

Eluent Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: S-impurities W-impurities Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 31

Inline Dil. Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 32

Feed Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Product Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 33

Inline Dil. Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 34

Eluent Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: Cycle complete , start next cycle Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 35

Eluent Eluent Step 1: elute waste Step 2: recycle overlap Step 3: elute product feed column Step 4: W-impurities S-impurities Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 36

Continuous Countercurrent Chromatography for three Stream Purifications MCSGP

Application of MCSGP: product classes
Small molecules Pharma Synthetic peptides, chiral molecules, macrolides Antibiotics Complex API Nutraceuticals/Food Fatty acids, Flavonoids, Polyphenols, Sweeteners Industrial biotech Fatty acids, monomers, organic acids Chemical intermediates Metals (REE) Natural extracts Proteins Recombinant bio-pharmaceuticals Monoclonal antibodies (mAbs) Antibody capture with CaptureSMB Antibody polish with MCSGP Aggregate removal 2nd generation products Biosimilars Antibody isoforms Bispecific antibodies PEGylated and conjugated proteins Blood plasma products Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

mAb charge isoform separation (Cation Exchange)

Example : varying mAb profiles
Feed Product (variable isoform content) (Contichrom-purified) Avastin® (Bevacizumab) Herceptin® (Trastuzumab) Ref: T. Müller-Späth, M. Krättli, L. Aumann, G. Ströhlein, M. Morbidelli: Increasing the Activity of Monoclonal Antibody Therapeutics by Continuous Chromatography (MCSGP), Biotechnology and Bioengineering, Volume 107, Issue 4, pages , 1 November 2010 Erbitux® (Cetuximab) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Comparison of Batch and MCSGP chromatography
Herceptin: Yield-Purity trade-off: Inherent to batch chromatography, less important for MCSGP Prod: 0.12 g/L/h Prod: 0.12 g/L/h MCSGP Batch trade-off Prod: 0.03 g/L/h Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

MCSGP operation - stability
Robustness of process against feed quality variations Feed spiked with mAb isoforms Blue: Regular Feed Red: High W feed Feed Product Feed Blue: Regular Feed Red: Spiked feed Blue: Regular Feed Red: Spiked feed Purified with same MCSGP process conditions MCSGP product purity: Not affected by change of feed. Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 42

Example: Biobetter mAb «Herceptin»
Originator mAb product «Herceptin» contains 7 isoforms with different activities (10%-150%) Using MCSGP, a homogeneous biobetter product has been isolated with high yield and purity, having 140% activity Potential for a Biobetter „Herceptin“ with lower dosing and better safety profile shown Isoform heterogeneity applies to all therapeutic mAbs Activity of Herceptin isoforms 140% 100% 12-30% Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 43

Bispecific antibody separation (Cation Exchange)

(Representative analytical chromatogram (CIEX) of the clarified harvest) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

delivers high purity >99.5%
MCSGP performance 2-column MCSGP: delivers high purity >99.5% increases yield by 50% - batch yield: 37% - MCSGP yield: 87% batch +50% yield Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

α-1-Antitrypsin purification from human plasma (Cation exchange)

α-1-Antitrypsin purification from human plasma
– %B HSA AAT Buffer Peaks IgG Analytical AIEX chromatogram Analytical results confirmed by ELISA

MCSGP Weak (IgG, HSA) Product (AAT) Strong Impurities Purity [%] Yield [%] Batch (max. P) 76.66 33.35 Batch (max. Y) 65 86.47 MCSGP 76.08 86.74

PEGylated protein separation (Anion Exchange)

Purification of PEGylated proteins
Constraints: Low yield of desired species at expensive production step using batch chromatography MCSGP provides 50% higher yield and purity with 5x higher throughput Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Purification of PEGylated proteins
MCSGP provides 50% higher yield with 5x higher throughput Analytical SEC of feed and MCSGP product MCSGP: +10% purity MCSGP: +30% yield Batch chromatography Prep. AIEX Batch elution of feed (load 4.3 g/L) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Peptide purification I (Reverse phase)

Polypetide purification
Peptide, ca. 46% pure, hundreds of unknown impurities Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Purification Result - Polypeptide

Purification Result - Productivity
Joint project with Novartis Pharma on Calcitonin: Productivity [g/L/h] factor 25 Yield for constant purity [%] Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Peptide purification II (Reverse phase)

Feed and representative batch material
Comparison of feed and representative batch chromatography pool from BMS Feed material – red BMS batch chromatography pool – blue A215 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Comparison of Batch and MCSGP
Overview of results: Analytical chromatography Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Overview of results: Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Overview of results: Purity-Yield chart. Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Fatty acid Ethyl Ester separation (Reverse phase)

MCSGP for -3 fatty acid ethyl ester production (EPA-EE)
Perform analytical RP-HPLC batch chromatography Feed purity 74%, target purity >97% (generic fish oil feed purchased from TCI Europe N.V.) Main impurity Docosahexaeonic acid ethyl ester (DHA-EE) EPA-EE DHA-EE Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Define sections of pure product extraction (red) and product-containing impurity recycling (blue + green) based on prior analytical purity measurements using the Contichrom® Software Wizard 15.8min min min waste recycle product recycle waste Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Online evaluation of MCSGP experiment: Cycle overlay can be used to determine if run has reached cyclic steady state and shows a consistent pattern Example: Overlay of 5 cycles: Exact matching of product profiles in (blue) window.  Cyclic steady state: Product of constant quality and concentration is withdrawn  Product consistency shown product elution Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Result chromatograms Overlay of analytical reversed phase chromatograms of feed and fractions from MCSGP Feed: Ratio EPA/DHA= 4:1 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Process for production of > 97% purity EPA-EE developed based on reverse phase chromatography with Ethanol as solvent Resin & solvent cost reduction of 80% with respect to batch chromatography MCSGP (20 m resin) Batch (15 m resin) Improvement by MCSGP Purity [%] >97% Yield [%] 90% 36% + 250% Productivity (Throughput) [(g product)/(L resin)/(hr operation time)] 65 11 + 590% Solvent Consumption [L solvent/g product] 0.8 3.2 - 75% Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Multicolumn countercurrent chromatography with very selective stationary phases (eg, Protein A) Objective: Improve Capacity Utilization

Resin capacity utilization in Batch vs. CaptureSMB
Feed conc. BTC X% DBC CaptureSMB Capacity gain Concentration [%] Batch Captured in DS column 1% DBC AmsphereTM Protein A is expected to perform very well in comparison to the benchmark product of GE due to its mass transfer properties L LX Load [g/Lresin] In twin column CaptureSMB the upstream column is loaded beyond its 1%DBC The flow through is captured by the downstream column The countercurrent sequential loading allows for a better capacity utilization and productivity while decreasing the buffer consumption Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Process Principle Batch Column Continuous Multicolumn
feed unused resin capacity elution feed fully loaded column Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Multicolumn Capture Processes: 4-col process
4-column process (4C-PCC): Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Multicolumn Capture Processes
3C-PCC principle presented by Genzyme (June 2012): Continuous feed with the same flow rate in all phases Biotechnology and Bioengineering, Vol. 109, No. 12, December, 2012 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

CaptureSMB Process schematic
Batchstep IC step Cyclic steady state Startup Switch 1 Switch 2 Shutdown Feed Waste 1 2 Elution CIP Equilib. P Wash IC step Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Continuous Countercurrent Chromatography
in three stream purifications breaks the batch trade-off in capture applications increases capacity utilization yield alternatives ? purity Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

….and all of this comes on top of the classical advantages of continuous over batch operation already well established in various industries Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Materials and methods Feed: clarified cell culture harvest containing IgG1 at a 1.2 g/L Column dimensions: Batch: ID 0.5 cm x BH 20 cm CaptureSMB: ID 0.5 cm x BH 10 cm (2 alternating columns) Resin: AmsphereTM Protein A JWT203 Chromatographic equipment: Contichrom® Lab-10 Analytical methods: mAb concentration: Prot-A HPLC (Poros® A-20), IEC HPLC (Tosoh SP STAT) Aggregate content: SEC HPLC (Tosoh TSK-Gel G3000SWXL) Host cell protein clearance: ELISA (Cygnus CHO-HCP 3rd generation) DNA clearance: Quant-iTTM PicoGreen® dsDNA (Life Technologies) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Results: Batch vs. CaptureSMB
Chromatograms for batch and CaptureSMB IgG purification using AmsphereTM Protein A JWT203 and Contichrom® Lab-10 Feed Wash Elution CIP Elution CIP Feed Wash Elution CIP Feed Wash Batch step 1 Interconnected step1 Batch step 2 Interconnected step 2 one UV detector at each column outlet Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Performance comparison: Batch vs. CaptureSMB
+11% +26% +23% +41% +35% -28% +32% -19% -13% +34% -9% +26% CaptureSMB process shows significant advantages in terms of loading (capacity utilization), productivity and buffer consumption in comparison to batch processes. Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Economic evaluation: scale-up model for resin costs
PoC Phase III Commercial Products per year [-] 8 2 1 Product per harvest [kg] 4 10 24 Fermenter harvest size [L] 2000 5000 12000 Product concentration [g/L] Harvests per year Effective production per year [Kg] 32 80 192 Harvest processing time [h] Resin lifetime 1 harvest 4 harvests 200 cycles Resin exchange after max. [Year] n.a. Resin costs AmsphereTM [US$/L] 13000* Significant resin cost savings (-25%) achieved by CaptureSMB Batch CaptureSMB * Indicative price only meant for simulation purposes Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Summary Comparison of CaptureSMB and batch process for 1g/L IgG1 capture case: Comparable product quality in terms of DNA, HCP and aggregates Higher loading (up to +40%) and productivity (up to +35%) Decreased buffer consumption (up to -25%) Higher product concentration (up to + 40%) In comparison with 3-/4-column cyclic processes, the twin-column CaptureSMB process requires less hardware complexity and has less risk of failure Economic evaluation using different scale-up scenarios showed synergistic cost saving effects of AmsphereTM JWT203 and CaptureSMB: Up to 25% cost savings (0.5M US$ annually) in PoC scenario compared to batch chromatography Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Conclusions and Outlook

Chromatography Process Classification
Continuous Periodic Carousel-Multicolumn chromatography Tandem-Capture Batch chromatography Fixed bed BioSMB, 3C-PCC (e.g. mAb Capture) 4-zone SMB (2-fractions, e.g. for enantiomers) pCAC (cont. annular chrom), cross-current CaptureSMB (e.g. mAb Capture) (Simulated) moving bed, Countercurrent MCSGP (3-fractions, e.g. for aggregate/fragment/mAb separation) Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Which kind of separation challenges exist?
Purification challenge Capture step (large selectivities) Sharp breakthrough curve Batch Slow loading Diffuse breakthrough curve Fast loading CaptureSMB Polish step Ternary separation Very difficult separation N-Rich Difficult separation MCSGP Baseline separated Binary separation SMB Decision tree for optimal choice of processes for any application All of these processes can be used with one single equipment Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Why 2 column processes are robust
More columns need more hardware, creating significantly more complexity and risk for component breakdown More columns mean more pumps and valves: the equipment gets more expensive and more complex! Original MCSGP setup with 8-columns Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

mAb (clarified harvest)
Outlook Most benefits of countercurrent chromatography can be realized with only 2 columns, keeping a reasonable level of equipment complexity Twin-column countercurrent chromatography processes are versatile and well suited for integrated bio-manufacturing Cyclic, countercurrent operation of capture and polishing steps Example process: mAb (clarified harvest) Pure mAb CaptureSMB® mode Protein A resin MCSGP mode CIEX resin or MM resin Tandem mode AIEX or MM resin Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Appendix Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Periodic upstream, periodic downstream
Operational need for continuous (feed) downstream process? Batch Periodic countercurrent Harvest clarification (Fed-) Batch upstream production DSP Downstream process: No need for constant feed flow rate, can use periodic process! Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Continuous upstream, continuous downstream?
Operational need for continuous (feed) process or periodic downstream process? Continuous DSP process Periodic DSP process Surge bag Cont. Clarifi-cation perfusion Continuous upstream production Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

BTC simulations using a lumped kinetic model
Parameter: qsat = 56.7 mg/ml, km= min-1 Experimental data fitting BTC predicted from model

Experimental conditions: Batch chromatography
Buffers: Method: Equilibration A 20 mM Phos, 150 mM NaCl, pH 7.5 Wash B 20 mM Phos, 1 M NaCl, pH 7.5 Elution C 50 mM Na-Cit, pH 3.2 CIP D 0.1 M NaOH Step CV [ml] Equilibration (A) 5 Load Wash-1 (A) Wash-2 (B) Wash-3 (A) Elution (C) CIP (D) 7.5 Re-Equi-1 (C) 2 Re-Equi-2 (A) 3 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

BTC simulations using a lumped kinetic model
Parameter: H= 4.69E3, qsat = 57 mg/ml, km= min-1 dax= cm Experimental data fitting BTC predicted from model

Internal concentration profiles: 3-Col process
Simulation parameters: lumped kinetic model Q= 0.84 ml/min, H= 4.69E3, qsat = 55 mg/ml, km= min-1

Economic evaluation: buffer consumption per year
PoC Phase III Commercial Product per harvest [kg] 4 10 24 Fermenter harvest size [L] 2000 5000 12000 Product concentration [g/L] 2 Harvests per year [-] 8 Effective production per year [Kg] 32 80 192 Harvest processing time [h] Resin lifetime 1 harvest 4 harvests 200 cycles Resin exchange after max. [Year] n.a. 1 Resin costs AmsphereTM [US$/L] 13000 Resin costs Agarose 17500 Significant buffer consumption savings achieved using Amsphere JWT 203 and CaptureSMB Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

Multicolumn Continuous Countercurrent Chromatography

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