1. Institute for Chemical
and Bioengineering
Multicolumn Continuous Countercurrent
Chromatography
Massimo Morbidelli
Institute for Chemical and Bioengineering, ETH Zurich, Switzerland
Integrated Continuous Biomanufacturing 2013,
20th – 24th Oct, Barcelona
2. Institute for Chemical
and Bioengineering
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
2
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7
Selective adsorption leads to
different elution velocities: select switch times
Features:
Linear gradients
Three fraction separations
Batch Chromatography
slow component
liquid
flow
chromatographic column
fast
component
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8
Continuous Countercurrent Chromatography
Selective adsorption leads to
different elution velocities: select solid speed
liquid
flow
solid flow
?
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5. Institute for Chemical
and Bioengineering
9
Simulated Moving Bed Chromatography
2
2
The SMB scheme:
Extract
(strongly adsorbing)
Feed
Raffinate
(early eluting)
4
4
1
1
3
3
Eluent
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6. Institute for Chemical
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Batch versus SMB performance
Separation of a pharmaceutical intermediate racemate
mixture on a chiral stationary phase (CSP)1
1 J.Chrom A 1006 (1-2): 267-280, 2003
0
0.5
1
1.5
2
2.5
3
Solvent requirement Productivity
HPLC Batch
SMB
Eluent need [L/g]
-80%
8x
Productivity [g/ kg/min]
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7. Institute for Chemical
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Typical bio-purification problem
Example: mAb purification from cell culture supernatant
typical chromatogram for mAb elution on cation-exchanger:
mAb
HCPs
fragments
aggregates
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 12
8. Institute for Chemical
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Purification challenge
Generic purification problem:
separate into 3 fractions
#2: mAb
#1: early eluting impurities #3: late eluting impurities
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9. Institute for Chemical
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Purification challenge
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 14
10. Institute for Chemical
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Purification challenge
in 3-fraction batch chromatography:
intrinsic trade-off between yield and purity!
Alternatives:
- Very Selective Stationary Phase (eg, Protein A)
- Continuous Countercurrent Chromatography (MCSGP)
purity
yield
alternatives ?
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 15
11. Institute for Chemical
and Bioengineering
Batch chromatography: SMB:
pulsed feed
multi-fraction separation
linear solvent gradients
low efficiency binary separation
step solvent gradients
continuous feed
counter-current operation
high efficiency
Combining batch and SMB
MCSGP (Multi-column Countercurrent Solvent Gradient Purification):
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 16
12. Institute for Chemical
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Principle 6 Column Purification unit
t
t
t t tF
H
P
L
inerts
c
1. Load // elute light
2. elute overlapping
product/light
3. elute product
4. elute overlapping
heavy/product
5. elute heavy
6. Receive overlapping
product/light
1
2
3
4
5 6
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Animation 6 Column MCSGP unit
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14. Institute for Chemical
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Contichrom® & MCSGP explained
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Continuous Countercurrent Chromatography
for three Stream Purifications
MCSGP
37
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16. Institute for Chemical
and Bioengineering
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
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mAb charge isoform separation
(Cation Exchange)
39
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Example : varying mAb profiles
Feed 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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 40
19. Institute for Chemical
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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
yield
_
Batch > 90% purity
Batch > 80% purity
MCSGP
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
MCSGP
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 41
20. Institute for Chemical
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MCSGP operation - stability
Robustness of process against feed quality variations
Feed spiked with mAb isoforms
Blue:
Regular
Feed
Red: High
W feed
Feed
Blue:
Regular
Feed
Red:
Spiked
feed
Blue:
Regular
Feed
Red:
Spiked
feed
Feed Product
MCSGP product purity: Not affected by change of feed.
Purified with
same MCSGP
process
conditions
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 42
21. Institute for Chemical
and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 43
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Bispecific antibody separation
(Cation Exchange)
44
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Purification challenge
45
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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%
46
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
batch +50% yield
25. Institute for Chemical
and Bioengineering
α-1-Antitrypsin purification from
human plasma
(Cation exchange)
47
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Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
α-1-Antitrypsin purification from human plasma
48
– A280
– %B
HSA
AAT
IgG Buffer
Peaks
Analytical results confirmed by ELISA
Analytical AIEX chromatogram
27. Institute for Chemical
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Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
α-1-Antitrypsin purification from human plasma
49
28. Institute for Chemical
and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
α-1-Antitrypsin purification from human plasma
50
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
29. Institute for Chemical
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PEGylated protein separation
(Anion Exchange)
51
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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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 52
31. Institute for Chemical
and Bioengineering
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)
MCSGP: +10% purity
MCSGP:
+30% yield
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 53
32. Institute for Chemical
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Peptide purification I
(Reverse phase)
54
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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:
Productivity
[g/L/h]
Yield for constant purity [%]
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 59
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Peptide purification II
(Reverse phase)
60
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Feed and representative batch material
Comparison of feed and representative batch chromatography pool
from BMS
A215
Feed material – red
BMS batch chromatography pool – blue
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Comparison of Batch and MCSGP
Overview of results: Analytical chromatography
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41. Institute for Chemical
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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
Purity
[%]
Yield [%]
A215
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 63
42. Institute for Chemical
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Comparison of Batch and MCSGP
Overview of results: Purity-Yield chart.
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
Purity
[%]
Yield [%]
Batch
MCSGP
Prod= 28-31 g/L/h
S.C. =0.9-1.0 L/g
conc.P = 8.4-9.3 g/L
Prod= 14 g/L/h
S.C.=0.7 L/g
conc. P = 3.3 g/L
Prod= 3 g/L/h
S.C.=3.5 L/g
conc. P = 8.2 g/L
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 64
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Fatty acid Ethyl Ester separation
(Reverse phase)
65
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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)
66
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EPA-EE DHA-EE
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0
20
40
60
80
100
120
140
160
14 16 18 20 22 24
concentration
(normalized)
Time [min]
Feed
Product
W-fraction
S-fraction
EPA-EE (> 97% pure)
DHA-EE
Impurity
FA-EE
MCSGP for -3 fatty acid ethyl ester production (EPA-EE)
Result chromatograms
69
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Overlay of analytical reversed
phase chromatograms of feed
and fractions from MCSGP
Feed: Ratio EPA/DHA= 4:1
46. Institute for Chemical
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MCSGP for -3 fatty acid ethyl ester production (EPA-EE)
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% >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 70
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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
71
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Process Principle
Batch Column
Continuous Multicolumn
feed
unused resin
capacity
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)
elu
wash
(ups)
load
(ups)
Load
(ds)
CIP
wash
load wash
(ds)
elu wash
(ups)
load
(ups)
Load
(ds)
CIP wash
load
wash
(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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Batch
step
IC
step
Cyclic
steady
state
Startup
Switch 1
Switch 2
Shutdown
Feed
Waste
1 2
Elution
CIP
Equilib.
Waste
1 2
Feed
Waste
P
1 2
Feed
Wash
Waste
IC
step
Elution
CIP
Equilib.
Waste
2
1
Feed
Waste
P
Feed
Waste
1 2
Batch
step
IC
step
Batch
step
Elution
CIP
Equilib.
1
Waste
P
Elution
CIP
Equilib.
2
Waste
P
CaptureSMB Process schematic
76
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52. Institute for Chemical
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Continuous Countercurrent Chromatography
in three stream purifications breaks the batch trade-off
in capture applications increases capacity utilization
purity
yield
alternatives ?
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 77
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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
78
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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
83
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Conclusions and Outlook
84
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Chromatography Process Classification
85
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
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Purification
challenge
Capture step
(large
selectivities)
Sharp
breakthrough
curve
Batch
Slow loading
Diffuse
breakthrough
curve
CaptureSMB
Fast 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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 86
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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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 87
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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®
mode
Protein A resin
MCSGP mode
CIEX resin or
MM resin
mAb
(clarified
harvest)
Pure
mAb
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 88
Tandem mode
AIEX or MM
resin
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Appendix
89
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Periodic upstream, periodic downstream
Operational need for continuous (feed) downstream
process?
90
(Fed-) Batch
upstream
production
Harvest clarification
Downstream process: No need for constant
feed flow rate, can use periodic process!
Batch
Periodic countercurrent
DSP
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Continuous upstream, continuous downstream?
Operational need for continuous (feed) process or periodic
downstream process?
91
Continuous upstream production
perfusion Cont.
Clarifi-
cation
Continuous DSP process
Periodic DSP process
Surge bag
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BTC simulations using a lumped kinetic model
92
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 7.5
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
93
Step CV [ml]
Equilibration (A) 5
Load
Wash-1 (A) 5
Wash-2 (B) 5
Wash-3 (A) 5
Elution (C) 5
CIP (D) 7.5
Re-Equi-1 (C) 2
Re-Equi-2 (A) 3
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BTC simulations using a lumped kinetic model
94
Experimental data fitting
BTC predicted from model
Parameter: H= 4.69E3,
qsat = 57 mg/ml, km= 0.077 min-1 dax= 42.28 cm
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Internal concentration profiles: 3-Col process
95
2 4 6 8 10
0
1
2
c
[mg/ml]
Column 1: Regenerating
2 4 6 8 10
0
20
40
60
80
Column Position [cm]
q
[mg/ml]
2 4 6 8 10
0
1
2
Column 2: Loading
2 4 6 8 10
0
20
40
60
80
Column Position [cm]
2 4 6 8 10
0
1
2
Column 3: FT uptake
2 4 6 8 10
0
20
40
60
80
Column Position [cm]
Simulation parameters: lumped kinetic model
Q= 0.84 ml/min, H= 4.69E3, qsat = 55 mg/ml, km= 0.077 min-1
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Economic evaluation: buffer consumption per year
96
Significant buffer consumption
savings achieved using
Amsphere JWT 203 and
CaptureSMB
PoC Phase III Commercial
Product per harvest [kg] 4 10 24
Fermenter harvest size [L] 2000 5000 12000
Product concentration [g/L] 2 2 2
Harvests per year [-] 8 8 8
Effective production per year [Kg] 32 80 192
Harvest processing time [h] 24 24 24
Resin lifetime [-] 1 harvest 4 harvests 200 cycles
Resin exchange after max. [Year] n.a. n.a. 1
Resin costs AmsphereTM
[US$/L] 13000 13000 13000
Resin costs Agarose [US$/L] 17500 17500 17500
0
50
100
150
200
250
PoC Ph III Comm.
[1000
L]
Buffer consumption per year
(300 cm/h)
0
50
100
150
200
250
PoC Ph III Comm.
[1000
L]
Buffer consumption per year
(600 cm/h)
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