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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
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 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
8
Continuous Countercurrent Chromatography Selective adsorption leads to different elution velocities: select solid speed
liquid flow
solid flow
?
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
10
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 BatchSMB
Eluent need [L/g]
-80%
8x
Productivity [g/ kg/min]
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
Purification challenge Generic purification problem:
separate into 3 fractions
#2: mAb
#1: early eluting impurities #3: late eluting impurities
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 13
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
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)
process
purity
yield
alternatives ?
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 15
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
Institute for Chemical and Bioengineering
Principle 6 Column Purification unit
tδ tβ tγ tα 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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 19
Institute for Chemical and Bioengineering
Animation 6 Column MCSGP unit
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 20
Institute for Chemical and Bioengineering
Contichrom® & MCSGP explained
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 24
Institute for Chemical and Bioengineering
Continuous Countercurrent Chromatography for three Stream Purifications
MCSGP
37 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
mAb charge isoform separation (Cation Exchange)
39 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
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
MCSGP
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 41
Institute for Chemical and Bioengineering
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
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
Institute for Chemical and Bioengineering
Bispecific antibody separation (Cation Exchange)
44 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Purification challenge
45 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
(Representative analytical chromatogram (CIEX) of the clarified harvest)
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
α-1-Antitrypsin purification from human plasma
(Cation exchange)
47 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
α-1-Antitrypsin purification from human plasma
49
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
Institute for Chemical and Bioengineering
PEGylated protein separation (Anion Exchange)
51 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
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
Institute for Chemical and Bioengineering
Peptide purification I (Reverse phase)
54 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Polypetide purification
Peptide, ca. 46% pure, hundreds of unknown impurities
P
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 55
Institute for Chemical and Bioengineering
Purification Result - Polypeptide
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 56
Institute for Chemical and Bioengineering
Purification Result - Polypeptide
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 57
Institute for Chemical and Bioengineering
Purification Result - Polypeptide
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 58
Institute for Chemical and Bioengineering
Purification Result - Productivity
factor 25
Joint project with Novartis Pharma on Calcitonin:
P
rodu
ctiv
ity [g
/L/h
]
Yield for constant purity [%]
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 59
Institute for Chemical and Bioengineering
Peptide purification II (Reverse phase)
60 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Feed and representative batch material Comparison of feed and representative batch chromatography pool
from BMS
A215
Feed material – red BMS batch chromatography pool – blue
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 61
Institute for Chemical and Bioengineering
Comparison of Batch and MCSGP Overview of results: Analytical chromatography
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 62
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 63
Institute for Chemical and Bioengineering
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
Purit
y [%
]
Yield [%]
Batch
MCSGP
Prod = 28-31 g/L/hS.C. =0.9-1.0 L/gconc. P = 8.4-9.3 g/L
Prod = 14 g/L/hS.C. =0.7 L/gconc. P = 3.3 g/L
Prod = 3 g/L/hS.C. =3.5 L/gconc. P = 8.2 g/L
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 64
Institute for Chemical and Bioengineering
Fatty acid Ethyl Ester separation (Reverse phase)
65 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
MCSGP for ω-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 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
EPA-EE DHA-EE
Institute for Chemical and Bioengineering
0
20
40
60
80
100
120
140
160
14 16 18 20 22 24
conc
entr
atio
n (n
orm
alize
d)
Time [min]
Feed
Product
W-fraction
S-fraction
EPA-EE (> 97% pure)
DHA-EEImpurity FA-EE
MCSGP for ω-3 fatty acid ethyl ester production (EPA-EE)
Result chromatograms
69 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Overlay of analytical reversed phase chromatograms of feed and fractions from MCSGP Feed: Ratio EPA/DHA= 4:1
Institute for Chemical and Bioengineering
MCSGP for ω-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 µm resin)
Batch (15 µm 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
Institute for Chemical and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Multicolumn countercurrent chromatography with very selective stationary phases (eg, Protein A)
Objective: Improve Capacity Utilization
71
Institute for Chemical and Bioengineering
Process Principle
Batch Column
Continuous Multicolumn
feed
unused resin capacity
feed
fully loaded column
elution
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 73
Institute for Chemical and Bioengineering
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):
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 74
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 75
Institute for Chemical and Bioengineering
Batchstep
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
Batchstep
IC step
Batchstep
Elution CIP
Equilib. 1
Waste
P Elution
CIP Equilib.
2
Waste
P
CaptureSMB Process schematic
76 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
….and all of this comes on top of the classical advantages of continuous over batch operation already
well established in various industries
78 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Conclusions and Outlook
84 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
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
Institute for Chemical and Bioengineering
Appendix
89 Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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
Institute for Chemical and Bioengineering
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Institute for Chemical and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
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
Institute for Chemical and Bioengineering
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli
Internal concentration profiles: 3-Col process
95
2 4 6 8 100
1
2
c [m
g/m
l]
Column 1: Regenerating
2 4 6 8 10020406080
Column Position [cm]
q [m
g/m
l]
2 4 6 8 100
1
2
Column 2: Loading
2 4 6 8 10020406080
Column Position [cm]
2 4 6 8 100
1
2
Column 3: FT uptake
2 4 6 8 10020406080
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
Institute for Chemical and Bioengineering
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.
[100
0 L]
Buffer consumption per year (300 cm/h)
0
50
100
150
200
250
PoC Ph III Comm.
[100
0 L]
Buffer consumption per year (600 cm/h)
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli