1-Morbidelli-131014 Presentation Barcelona MM

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
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
Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 2

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Batch Chromatography
Selective adsorption leads to fast
different elution velocities: select switch times 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 4
1
3
1
3
2 2
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
2.5
2
HPLC Batch 8x
1.5 SMB
1
-80%
0.5
0
Eluent needrequirement
Solvent [L/g] Productivity
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
aggregates
fragments

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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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in 3-fraction batch chromatography:

intrinsic trade-off between yield and purity!
high yield, low purity high purity, low yield

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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

1. Load // elute light
2. elute overlapping
product/light
3. elute product
4. elute overlapping
heavy/product
5. elute heavy
6. Receive overlapping
5 4 3 2 1 6 product/light
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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for three Stream Purifications
MCSGP

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Application of MCSGP: product classes
Small molecules Proteins
Pharma Recombinant bio-

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

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mAb charge isoform separation

(Cation Exchange)

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Example : varying mAb profiles

Feed Product
(variable isoform content) (Contichrom-purified)
Avastin
(Bevacizumab)
Herceptin
(Trastuzumab)
Ref: T. Mller-Spth, M. Krttli, L.

Aumann, G. Strhlein, M.
Erbitux Morbidelli: Increasing the Activity
of Monoclonal Antibody
(Cetuximab) Therapeutics by Continuous
Chromatography (MCSGP),
Biotechnology and
Bioengineering, Volume 107,
Issue 4, pages 652-662, 1
November 2010

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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%
70.0%
MCSGP
_
60.0%
yield
50.0%
40.0%
30.0%
Batch > 90% purity
20.0% Batch > 80% purity
10.0% MCSGP Prod: 0.03 g/L/h
0.0%
78.0% 80.0% 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
Feed
Blue: Product
Regular Feed
Blue:
Feed Blue:
Red: High
Regular Regular
W feed
Feed Purified with
Feed
Red: same MCSGP
Red:
Spiked process
Spiked
feed conditions
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
Activity of Herceptin isoforms
with different activities (10%-150%)
Using MCSGP, a homogeneous
140%
biobetter product has been isolated
with high yield and purity, having 100%
12-30%
140% activity
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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(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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-1-Antitrypsin purification from

human plasma
(Cation exchange)

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-1-Antitrypsin purification from human plasma
A280
HSA %B
IgG AAT Buffer

Peaks
Analytical AIEX chromatogram

Analytical results confirmed by ELISA
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MCSGP
Weak Product Strong

(IgG, HSA) (AAT) Impurities
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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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
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

Joint project with Novartis Pharma on Calcitonin:
Productivity [g/L/h]
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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Overview of results:
99.0
98.5
98.0
Purity [%]
97.5
97.0
96.5
96.0
0 10 20 30 40 50 60 70 80 90 100
A215 Yield [%]

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Overview of results: Purity-Yield chart.
99.0
Prod = 28-31 g/L/h
98.5 S.C. =0.9-1.0 L/g
conc. P = 8.4-9.3 g/L
Prod = 3 g/L/h
98.0 S.C. =3.5 L/g
conc. P = 8.2 g/L
Purity [%]
97.5
97.0
Batch Prod = 14 g/L/h
96.5 MCSGP S.C. =0.7 L/g
conc. P = 3.3 g/L
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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Result chromatograms
160
Feed
140 Product
W-fraction EPA-EE (> 97% pure)
120 S-fraction
concentration (normalized)
Overlay of analytical reversed

100 phase chromatograms of feed
and fractions from MCSGP
80
Feed: Ratio EPA/DHA= 4:1
60
Impurity DHA-EE
40 FA-EE
20
0
14 16 18 20 22 24
Time [min]

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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 Batch Improvement by

(20 m (15 m MCSGP
resin) resin)
Purity [%] >97% >97%
Yield [%] 90% 36% + 250%
Productivity (Throughput) 65 11 + 590%
[(g product)/(L resin)/(hr operation time)]
Solvent Consumption 0.8 3.2 - 75%

[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 elu wash
Switch 1 (ds) (ups)
load Load CIP wash

Switch 2 (ups) (ds)
Switch 3 wash load wash elu

(ups) (ds)
Switch 4 wash load Load CIP

(ups) (ds)
Switch 5 elu wash load wash

(ups) (ds)
Switch 6 CIP wash load Load

(ups) (ds)
wash elu wash load

Switch 7
(ds) (ups)
Load CIP wash load

Switch 8 (ds) (ups)

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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 1 2
Feed
step
Waste
Batch Elution
step 1 CIP 2 P
Feed Equilib.
Switch 1 Waste
Waste
IC
step 1 2
Feed
Wash
Cyclic
Waste
steady
Batch Elution state
CIP 1 P 2
step Equilib. Feed
Switch 2 Waste
Waste
IC 2
1
step Feed
Elution Elution
Batch
Shutdown step
CIP
Equilib.
1 P
CIP
Equilib.
2 P
Waste Waste

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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 Periodic
Carousel-
Fixed bed Multicolumn Batch
chromatography chromatography
Tandem-Capture
BioSMB, 3C-PCC CaptureSMB

(e.g. mAb Capture) (e.g. mAb Capture)
(Simulated)
4-zone SMB
moving bed, (2-fractions, e.g. for MCSGP
Countercurrent enantiomers) (3-fractions, e.g. for
aggregate/fragment/mAb
pCAC (cont. annular separation)
chrom), cross-current

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Which kind of separation challenges exist?

Decision tree for optimal choice of processes for any application
Sharp Batch
breakthrough
curve Slow loading
(large
Capture step
selectivities) Diffuse CaptureSMB
breakthrough
curve Fast loading
Very difficult
separation N-Rich
Purification
challenge
Ternary Difficult
separation separation MCSGP
Baseline
separated Batch
Polish step
Difficult
separation SMB
Binary
separation
Baseline
separated Batch
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 Pure
harvest) mAb
CaptureSMB MCSGP mode Tandem mode

mode CIEX resin or AIEX or MM
Protein A resin MM resin resin

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Appendix

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Periodic upstream, periodic downstream

Operational need for continuous (feed) downstream
process?
Batch
Harvest clarification Periodic countercurrent

(Fed-) Batch DSP
upstream
production
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 Cont.
Clarifi- Surge bag
cation
Continuous upstream production
Periodic DSP process

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BTC simulations using a lumped kinetic model

Parameter: qsat = 56.7 mg/ml,
Experimental data fitting km= 0.051 min-1
BTC predicted from model

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Experimental conditions: Batch chromatography

Buffers:
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
Method:
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

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

Column 1: Regenerating Column 2: Loading Column 3: FT uptake
2 2 2
c [mg/ml]
1 1 1
0 0 0
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
80 80 80
q [mg/ml]
60 60 60
40 40 40
20 20 20
0 0 0
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
Column Position [cm] Column Position [cm] 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
Significant buffer consumption
Product per harvest [kg] 4 10 24
Fermenter harvest size [L] 2000 5000 12000 savings achieved using
Product concentration [g/L] 2 2 2 Amsphere JWT 203 and
Harvests per year [-] 8 8 8 CaptureSMB
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$/L] 13000 13000 13000
Resin costs Agarose [US$/L] 17500 17500 17500
Buffer consumption per year Buffer consumption per year

(300 cm/h) (600 cm/h)
250 250
200 200
[1000 L]
[1000 L]
150 150
100 100
50 50
0 0
PoC Ph III Comm. PoC Ph III Comm.

1-Morbidelli-131014 Presentation Barcelona MM

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Institute for Chemical

Multicolumn Continuous Countercurrent

Institute for Chemical and Bioengineering, ETH Zurich, Switzerland

Process evolution: from batch to multicolumn simulated

Countercurrent Chromatography for three stream

Countercurrent Chromatography for highly selective

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 2

Continuous Countercurrent Chromatography

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 8

Simulated Moving Bed Chromatography

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 9

Batch versus SMB performance

Typical bio-purification problem

Example: mAb purification from cell culture supernatant

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 12

#1: early eluting impurities #3: late eluting impurities

in 3-fraction batch chromatography:

high yield, low purity high purity, low yield

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 14

Combining batch and SMB

multi-fraction separation continuous feed

pulsed feed high efficiency

MCSGP (Multi-column Countercurrent Solvent Gradient Purification):

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 16

Principle 6 Column Purification unit

Animation 6 Column MCSGP unit

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 20

Contichrom & MCSGP explained

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 24

Continuous Countercurrent Chromatography

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 37

Application of MCSGP: product classes

Small molecules Proteins

Pharma Recombinant bio-

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli

mAb charge isoform separation

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 39

Example : varying mAb profiles

Ref: T. Mller-Spth, M. Krttli, L.

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 40

Comparison of Batch and MCSGP chromatography

MCSGP operation - stability

MCSGP product purity: Not affected by change of feed.

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 42

Example: Biobetter mAb Herceptin

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 43

Bispecific antibody separation

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 44

(Representative analytical chromatogram (CIEX) of the clarified harvest)

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 45

delivers high purity >99.5%

batch +50% yield

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 46

-1-Antitrypsin purification from

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 47

-1-Antitrypsin purification from human plasma

IgG AAT Buffer

Analytical AIEX chromatogram

-1-Antitrypsin purification from human plasma

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 49

-1-Antitrypsin purification from human plasma

Weak Product Strong

Purity [%] Yield [%]