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
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Batch Chromatography
 Selective adsorption leads to
different elution velocities: select switch times
fast
component
chromatographic column
liquid
flow
 Features:
 Linear gradients
 Three fraction separations
slow component
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Continuous Countercurrent Chromatography
Selective adsorption leads to
different elution velocities: select solid speed
?
liquid
flow
solid flow
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Simulated Moving Bed Chromatography
 The SMB scheme:
Eluent
Raffinate
(early eluting)
4
2
2
3
1
4
1
3
Feed
Extract
(strongly adsorbing)
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Batch versus SMB performance
 Separation of a pharmaceutical intermediate racemate
mixture on a chiral stationary phase (CSP)1
3
2.5
2
HPLC Batch
SMB
1.5
1
8x
-80%
0.5
0
Solvent
Productivity
Eluent
needrequirement
[L/g] Productivity
[g/ kg/min]
1
J.Chrom A 1006 (1-2): 267-280, 2003
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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
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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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Purification challenge
 in 3-fraction batch chromatography:
intrinsic trade-off between yield and purity!
high yield, low purity
high purity, low yield
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Purification challenge
 in 3-fraction batch chromatography:
intrinsic trade-off between yield and purity!
yield
alternatives ?
purity
 Alternatives:
- Very Selective Stationary Phase (eg, Protein A)
- Continuous Countercurrent Chromatography (MCSGP)
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Combining batch and SMB
Batch chromatography:
SMB:
 multi-fraction separation
 continuous feed
 linear solvent gradients
 counter-current operation
 pulsed feed
 high efficiency
 low efficiency
 binary separation
 step solvent gradients
MCSGP (Multi-column Countercurrent Solvent Gradient Purification):
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Principle 6 Column Purification unit
3
4
5
2
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
6
1
L
P
H
c
inerts
t
t
t
t
tF
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Animation 6 Column MCSGP unit
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Contichrom® & MCSGP explained
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Continuous Countercurrent Chromatography
for three Stream Purifications
MCSGP
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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 biopharmaceuticals
• 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)
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Example : varying mAb profiles
Feed
(variable isoform content)
Product
(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 652-662, 1
November 2010
Erbitux®
(Cetuximab)
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Comparison of Batch and MCSGP chromatography

Herceptin: Yield-Purity trade-off: Inherent to batch chromatography, less
important for MCSGP
100.0%
90.0%
Prod: 0.12 g/L/h
Prod: 0.12 g/L/h
80.0%
_
60.0%
yield
70.0%
50.0%
MCSGP
40.0%
30.0%
Batch > 90% purity
20.0%
Batch > 80% purity
10.0%
MCSGP
0.0%
78.0%
80.0%
Prod: 0.03 g/L/h
82.0%
84.0%
86.0%
88.0%
90.0%
92.0%
purity
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MCSGP operation - stability


Robustness of process against feed quality variations
Feed spiked with mAb isoforms
Blue:
Feed
Regular
Blue:
Feed
Red:
High
Regular
W feed
Feed
Red:
Spiked
feed
Product
Feed
Purified with
same MCSGP
process
conditions
Blue:
Regular
Feed
Red:
Spiked
feed
MCSGP product purity: Not affected by change of feed.
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Example: Biobetter mAb «Herceptin»

Originator mAb product
«Herceptin» contains 7 isoforms
with different activities (10%-150%)

Activity of Herceptin isoforms
Using MCSGP, a homogeneous
140%
biobetter product has been isolated
with high yield and purity, having
100%
140% activity

12-30%
Potential for a Biobetter „Herceptin“
with lower dosing and better safety
profile shown

Isoform heterogeneity applies to all
therapeutic mAbs
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Bispecific antibody separation
(Cation Exchange)
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Purification challenge
(Representative analytical chromatogram (CIEX) of the clarified harvest)
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MCSGP performance
2-column MCSGP:
 delivers high purity >99.5%
batch
+50% yield
 increases yield by 50%
- batch yield: 37%
- MCSGP yield: 87%
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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
– %B
HSA
IgG
Buffer
Peaks
AAT
Analytical AIEX chromatogram
Analytical results confirmed by ELISA
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α-1-Antitrypsin purification from human plasma
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α-1-Antitrypsin purification from human plasma
MCSGP
Weak
Product
(IgG, HSA) (AAT)
Strong
Impurities
Purity [%]
Yield [%]
Batch (max. P) 76.66
33.35
Batch (max. Y) 65
86.47
MCSGP
86.74
76.08
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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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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
Prep. AIEX Batch elution of feed (load 4.3 g/L)
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Peptide purification I
(Reverse phase)
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Polypetide purification
P
Peptide, ca. 46% pure, hundreds of unknown impurities
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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
Productivity [g/L/h]
 Joint project with Novartis Pharma on Calcitonin:
factor 25
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
Feed material – red
BMS batch chromatography pool – blue
A215
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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:
99.0
98.5
Purity [%]
98.0
97.5
97.0
96.5
96.0
0
A215
10
20
30
40
50
60
70
80
90
100
Yield [%]
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Comparison of Batch and MCSGP
 Overview of results: Purity-Yield chart.
99.0
Prod = 28-31 g/L/h
S.C. =0.9-1.0 L/g
conc. P = 8.4-9.3 g/L
98.5
Prod = 3 g/L/h
S.C. =3.5 L/g
conc. P = 8.2 g/L
Purity [%]
98.0
97.5
97.0
Batch
96.5
Prod = 14 g/L/h
S.C. =0.7 L/g
conc. P = 3.3 g/L
MCSGP
96.0
0
10
20
30
40
50
60
70
80
90
100
Yield [%]
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Fatty acid Ethyl Ester separation
(Reverse phase)
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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)
EPA-EE
DHA-EE
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MCSGP for -3 fatty acid ethyl ester production (EPA-EE)
Result chromatograms
160
Feed
140
Product
EPA-EE (> 97% pure)
W-fraction
120
concentration (normalized)

S-fraction
Overlay of analytical reversed
phase chromatograms of feed
and fractions from MCSGP
100
80
Feed: Ratio EPA/DHA= 4:1
60
Impurity
FA-EE
40
DHA-EE
20
0
14
16
18
20
Time [min]
22
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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)
65
11
+ 590%
0.8
3.2
- 75%
[(g product)/(L resin)/(hr operation time)]
Solvent Consumption
[L solvent/g product]
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Multicolumn countercurrent chromatography with
very selective stationary phases (eg, Protein A)
Objective: Improve Capacity Utilization
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Process Principle
 Batch Column
feed
unused resin
capacity
 Continuous Multicolumn
elution
feed
fully loaded column
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Multicolumn Capture Processes: 4-col process
 4-column process (4C-PCC):
1
2
3
4
load
wash
(ds)
elu
wash
(ups)
Switch 2
load
(ups)
Load
(ds)
CIP
wash
Switch 3
wash
(ups)
load
wash
(ds)
elu
Switch 4
wash
load
(ups)
Load
(ds)
CIP
Switch 5
elu
wash
(ups)
load
wash
(ds)
Switch 6
CIP
wash
load
(ups)
Load
(ds)
Switch 7
wash
(ds)
elu
wash
(ups)
load
Switch 8
Load
(ds)
CIP
wash
load
(ups)
Switch 1
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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
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CaptureSMB Process schematic
Waste
Startup
IC
step
Batch
step
1
Feed
2
Waste
Feed
Elution
CIP
Equilib.
1
2
P
Switch 1
Waste
Waste
IC
step
Feed
Wash
2
1
Waste
Switch 2
Batch
step
Elution
CIP
Equilib.
1
P
Feed
2
Cyclic
steady
state
Waste
Waste
IC
step
Shutdown
Batch
step
1
Elution
CIP
Equilib.
1
Feed
Elution
CIP
Equilib.
P
2
2
Waste
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P
Waste
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Continuous Countercurrent Chromatography
 in three stream purifications breaks the batch trade-off
yield
alternatives ?
purity
 in capture applications increases capacity utilization
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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
Continuous
Fixed bed
CarouselMulticolumn
chromatography
Periodic
Batch
chromatography
Tandem-Capture
BioSMB, 3C-PCC
(e.g. mAb Capture)
(Simulated)
moving bed,
Countercurrent
CaptureSMB
(e.g. mAb Capture)
4-zone SMB
(2-fractions, e.g. for
enantiomers)
pCAC (cont. annular
MCSGP
(3-fractions, e.g. for
aggregate/fragment/mAb
separation)
chrom), cross-current
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Which kind of separation challenges exist?
Decision tree for optimal choice of processes for any application
Capture step
(large
selectivities)
Purification
challenge
Ternary
separation
Polish step
Sharp
breakthrough
curve
Slow loading
Diffuse
breakthrough
curve
CaptureSMB
Fast loading
Very difficult
separation
N-Rich
Difficult
separation
MCSGP
Baseline
separated
Batch
Difficult
separation
SMB
Baseline
separated
Batch
Batch
Binary
separation
All of these processes can be used with one single equipment
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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:
mAb
(clarified
harvest)
Pure
mAb
CaptureSMB®
mode
Protein A resin
MCSGP mode
CIEX resin or
MM resin
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?
Batch
Periodic countercurrent
Harvest clarification
(Fed-) Batch
upstream
production
DSP
Downstream process: No need for constant
feed flow rate, can use periodic process!
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Continuous upstream, continuous downstream?
 Operational need for continuous (feed) process or periodic
downstream process?
Continuous DSP process
perfusion
Continuous upstream production
Cont.
Clarification
Surge bag
Periodic DSP process
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BTC simulations using a lumped kinetic model
Experimental data fitting
Parameter: qsat = 56.7 mg/ml,
km= 0.051 min-1
BTC predicted from model
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Experimental conditions: Batch chromatography
 Buffers:
Equilibration
Wash
Elution
CIP
A
B
C
D
20 mM Phos, 150 mM NaCl, pH 7.5
20 mM Phos, 1 M NaCl, pH 7.5
50 mM Na-Cit, pH 3.2
0.1 M NaOH
 Method:
Step
Equilibration (A)
Load
Wash-1 (A)
Wash-2 (B)
Wash-3 (A)
Elution (C)
CIP (D)
Re-Equi-1 (C)
Re-Equi-2 (A)
CV [ml]
5
5
5
5
5
7.5
2
3
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BTC simulations using a lumped kinetic model
Parameter: H= 4.69E3,
qsat = 57 mg/ml, km= 0.077 min-1 dax= 42.28 cm
Experimental data fitting
BTC predicted from model
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Internal concentration profiles: 3-Col process
c [mg/ml]
Column 1: Regenerating
Column 3: FT uptake
2
2
2
1
1
1
0
q [mg/ml]
Column 2: Loading
2
4
6
8
10
0
2
4
6
8
10
0
80
80
80
60
60
60
40
40
40
20
20
20
0
2
4
6
8 10
Column Position [cm]
0
2
4
6
8 10
Column Position [cm]
0
2
4
6
8
10
2
4
6
8 10
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
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
TM
Resin costs Amsphere
[US$/L] 13000
13000
13000
Resin costs Agarose
250
[US$/L]
17500
17500
17500
Buffer consumption per year
(300 cm/h)
250
Buffer consumption per year
(600 cm/h)
200
[1000 L]
200
[1000 L]
Significant buffer consumption
savings achieved using
Amsphere JWT 203 and
CaptureSMB
150
100
150
100
50
50
0
0
PoC
Ph III
Comm.
PoC
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Ph III
Comm.
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