Institute for Chemical and Bioengineering Multicolumn Continuous Countercurrent Chromatography Massimo Morbidelli Institute for Chemical and Bioengineering, ETH Zurich, Switzerland Integrated Continuous Biomanufacturing 2013, 20 th – 24 th Oct, Barcelona
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Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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mAb charge isoform separation(Cation Exchange)
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Example : varying mAb profilesFeed Product
Erbitux®(Cetuximab)
Herceptin®(Trastuzumab)
Avastin®(Bevacizumab)
(variable isoform content) (Contichrom-purified)
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 652-662, 1 November 2010
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0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
70.0%
80.0%
90.0%
100.0%
78.0% 80.0% 82.0% 84.0% 86.0% 88.0% 90.0% 92.0%
purity
yiel
d
_
Batch > 90% purityBatch > 80% purityMCSGP
Herceptin: Yield-Purity trade-off: Inherent to batch chromatography, less important for MCSGP
Comparison of Batch and MCSGP chromatography
Prod: 0.03 g/L/h
Prod: 0.12 g/L/h Prod: 0.12 g/L/h
Batch trade-off
MCSGP
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MCSGP operation - stability Robustness of process against feed quality variations Feed spiked with mAb isoforms
Blue: Regular FeedRed: High W feed
FeedBlue: Regular FeedRed: Spiked feed
Blue: Regular FeedRed: Spiked feed
Feed Product
MCSGP product purity: Not affected by change of feed.
Purified with same MCSGP process conditions
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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
100%
140%
12-30%
Activity of Herceptin isoforms
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Bispecific antibody separation(Cation Exchange)
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Purification challenge
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(Representative analytical chromatogram (CIEX) of the clarified harvest)
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MCSGP performance
2-column MCSGP:
delivers high purity >99.5%
increases yield by 50%- batch yield: 37%- MCSGP yield: 87%
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batch +50% yield
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α-1-Antitrypsin purification from human plasma
(Cation exchange)
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α-1-Antitrypsin purification from human plasma
– A280
– %BHSA
AATIgG BufferPeaks
Analytical results confirmed by ELISAAnalytical AIEX chromatogram
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α-1-Antitrypsin purification from human plasma
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α-1-Antitrypsin purification from human plasma
MCSGP
Weak(IgG, HSA)
Product(AAT)
StrongImpurities
Purity [%] Yield [%]
Batch (max. P) 76.66 33.35
Batch (max. Y) 65 86.47
MCSGP 76.08 86.74
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PEGylated protein separation (Anion Exchange)
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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
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MCSGP provides 50% higher yield with 5x higher throughput
Purification of PEGylated proteins
Analytical SEC of feed and MCSGP product
Prep. AIEX Batch elution of feed (load 4.3 g/L)
Batch chromatography
MCSGP: +10% purity
MCSGP:+30% yield
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Peptide purification I(Reverse phase)
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Polypetide purification
Peptide, ca. 46% pure, hundreds of unknown impurities
P
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Purification Result - Polypeptide
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Purification Result - Polypeptide
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Purification Result - Polypeptide
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Purification Result - Productivity
factor 25
Joint project with Novartis Pharma on Calcitonin:P
rodu
ctiv
ity [g
/L/h
]
Yield for constant purity [%]
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Peptide purification II(Reverse phase)
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Feed and representative batch material Comparison of feed and representative batch chromatography pool
from BMS
A215
Feed material – redBMS batch chromatography pool – blue
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Comparison of Batch and MCSGP Overview of results: Analytical chromatography
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Comparison of Batch and MCSGP Overview of results:
96.0
96.5
97.0
97.5
98.0
98.5
99.0
0 10 20 30 40 50 60 70 80 90 100
Purit
y [%
]
Yield [%]A215
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Comparison of Batch and MCSGP Overview of results: Purity-Yield chart.
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Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Multicolumn countercurrent chromatography with very selective stationary phases (eg, Protein A)
Objective: Improve Capacity Utilization
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Process Principle
Batch Column
Continuous Multicolumn
feed
unused resincapacity
feed
fully loaded column
elution
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Multicolumn Capture Processes: 4-col process
Switch 1
Switch 2
Switch 3
Switch 4
Switch 5
Switch 6
Switch 7
Switch 8
load wash(ds)
elu wash(ups)
1 2 3 4
load(ups)
Load(ds)
CIP wash
load wash(ds)
eluwash(ups)
load(ups)
Load(ds)
CIPwash
load wash(ds)
elu wash(ups)
load(ups)
Load(ds)
CIP wash
loadwash(ds)
elu wash(ups)
load(ups)
Load(ds)
CIP wash
4-column process (4C-PCC):
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3C-PCC principle presented by Genzyme (June 2012): Continuous feed with the same flow rate in all phases
Multicolumn Capture Processes
Biotechnology and Bioengineering, Vol. 109, No. 12, December, 2012
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Batchstep
ICstep
Cyclic steady state
Startup
Switch 1
Switch 2
Shutdown
Feed
Waste
1 2
ElutionCIP
Equilib.
Waste
1 2Feed
Waste
P
1 2FeedWash
WasteIC
step
ElutionCIP
Equilib.
Waste
21Feed
Waste
P
Feed
Waste
1 2
Batchstep
IC step
Batchstep
ElutionCIP
Equilib.1
Waste
PElution
CIPEquilib.
2
Waste
P
CaptureSMB Process schematic
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Continuous Countercurrent Chromatography
in three stream purifications breaks the batch trade-off
in capture applications increases capacity utilization
purity
yieldalternatives ?
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….and all of this comes on top of the classical advantages of continuous over batch operation already
well established in various industries
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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
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Conclusions and Outlook
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Chromatography Process Classification
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Continuous Periodic
(Simulated) moving bed, Countercurrent
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)
MCSGP(3-fractions, e.g. for aggregate/fragment/mAb separation)
Carousel-Multicolumn chromatography
Tandem-Capture
Fixed bed Batch chromatography
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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Purificationchallenge
Capture step (large selectivities)
Sharp breakthrough
curve
BatchSlow loading
Diffuse breakthrough
curve
CaptureSMBFast loading
Polish step
Ternary separation
Very difficult separation N-Rich
Difficult separation MCSGP
Baseline separated Batch
Binary separation
Difficult separation SMB
Baseline separated Batch
Which kind of separation challenges exist?
All of these processes can be used with one single equipment
Decision tree for optimal choice of processes for any application
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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
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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:
CaptureSMB®
modeProtein A resin
MCSGP modeCIEX resin or
MM resin
mAb (clarified harvest)
Pure mAb
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Tandem mode AIEX or MM
resin
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Appendix
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Periodic upstream, periodic downstream Operational need for continuous (feed) downstream
process?
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(Fed-) Batch upstream production
Harvest clarification
Downstream process: No need for constant feed flow rate, can use periodic process!
Batch
Periodic countercurrentDSP
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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Continuous upstream, continuous downstream? Operational need for continuous (feed) process or periodic
downstream process?
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Continuous upstream production
perfusion Cont.Clarifi-cation
Continuous DSP process
Periodic DSP process
Surge bag
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Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
BTC simulations using a lumped kinetic model
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Experimental data fitting
BTC predicted from model
Parameter: qsat = 56.7 mg/ml, km= 0.051 min-1
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Buffers:
Method:
Experimental conditions: Batch chromatography
Equilibration A 20 mM Phos, 150 mM NaCl, pH 7.5Wash B 20 mM Phos, 1 M NaCl, pH 7.5