Institute for Chemical and Bioengineering Multicolumn Continuous Countercurrent Chromatography Massimo Morbidelli Institute for Chemical and Bioengineering,

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


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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

2Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli


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Selective adsorption leads todifferent elution velocities: select switch times

Features: Linear gradients Three fraction separations

Batch Chromatography

slow component

liquidflow

chromatographic column

fastcomponent

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli


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Continuous Countercurrent Chromatography

Selective adsorption leads todifferent elution velocities: select solid speed

liquidflow

solid flow

?



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9

Simulated Moving Bed Chromatography

22

The SMB scheme:

Extract(strongly adsorbing)

Feed

Raffinate(early eluting)

441

1

33

Eluent



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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 Batch

SMB

Eluent need [L/g]

-80%

8x

Productivity [g/ kg/min]



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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

fragmentsaggregates

Integrated Continuous Biomanufacturing 2013, Barcelona / Massimo Morbidelli 12


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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!

Alternatives:

- Very Selective Stationary Phase (eg, Protein A)

- Continuous Countercurrent Chromatography (MCSGP)

process

purity

yield

alternatives ?



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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):



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Principle 6 Column Purification unit

ttt t tF

HP

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

12345 6



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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 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)



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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

yie

ld

_

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

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

Feed

Blue: 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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(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%


batch +50% yield


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α-1-Antitrypsin purification from human plasma

(Cation exchange)



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– A280

– %BHSA

AATIgG BufferPeaks

Analytical results confirmed by ELISA

Analytical AIEX chromatogram


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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 - Productivity

factor 25

Joint project with Novartis Pharma on Calcitonin:

Pro

duct

ivity

[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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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

Pu

rity

[%

]

Yield [%]A215



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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

Pu

rity

[%

]

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



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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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0

20

40

60

80

100

120

140

160

14 16 18 20 22 24

conc

entr

ation

(nor

mal

ized

)

Time [min]

Feed

Product

W-fraction

S-fraction

EPA-EE (> 97% pure)

DHA-EEImpurity FA-EE

MCSGP for -3 fatty acid ethyl ester production (EPA-EE)

Result chromatograms


Overlay of analytical reversed phase chromatograms of feed and fractions from MCSGP

Feed: Ratio EPA/DHA= 4:1


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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%



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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 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

P

ElutionCIP

Equilib.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

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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Purificationchallenge

Capture step (large selectivities)

Sharp breakthrough

curve

BatchSlow 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



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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


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?

90

(Fed-) Batch upstream production

Harvest clarification

Downstream process: No need for constant feed flow rate, can use periodic process!

Batch

Periodic countercurrentDSP



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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 7.5Wash B 20 mM Phos, 1 M NaCl, pH 7.5

Elution C 50 mM Na-Cit, pH 3.2CIP D 0.1 M NaOH

93

Step CV [ml]Equilibration (A) 5

Load Wash-1 (A) 5Wash-2 (B) 5Wash-3 (A) 5Elution (C) 5

CIP (D) 7.5Re-Equi-1 (C) 2Re-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 8 100

1

2

c [

mg

/ml]

Column 1: Regenerating

2 4 6 8 100

20

40

60

80

Column Position [cm]

q [

mg

/ml]

2 4 6 8 100

1

2

Column 2: Loading

2 4 6 8 100

20

40

60

80


2 4 6 8 100

1

2

Column 3: FT uptake

2 4 6 8 100

20

40

60

80


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 12000Product concentration [g/L] 2 2 2

Harvests per year [-] 8 8 8Effective production per year [Kg] 32 80 192

Harvest processing time [h] 24 24 24Resin 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

PoC Ph III Comm.0

50

100

150

200

250

Buffer consumption per year (300 cm/h)

[100

0 L]

PoC Ph III Comm.0

50

100

150

200

250

Buffer consumption per year (600 cm/h)

[100

0 L]


Documents

Institute for Chemical and Bioengineering Multicolumn Continuous Countercurrent Chromatography Massimo Morbidelli Institute for Chemical and Bioengineering,