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Continuous Chromatography for Monoclonal Antibody Purification from Cell Culture Supernatant

Massimo MorbidelliInstitute for Chemical and Bioengineering, ETH Zurich, Switzerland

2September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli

Content1. MAb purification challenge

2. Introduction to continuous liquid chromatography

3. Continuous gradient chromatography (MCSGP)

4. MAb purification from cCCS using ion-exchange

5. MAB Variant Separation

6. Comparison of technologies


MAb provided by Merck-Serono: mAb contains variants

IEF: analyt. CIEX (Propac wCX):

1. MAb purification challenge

pI range of mAb variants:7.4-8.2


MAb obtained from Merck-Serono:

analyt. CIEX (Propac wCX):


Batch pools:

Red: CIEXBlue: Protein A

mAb fragments, early eluting in CIEX



analyt. SEC (Tosoh):


Red: Protein A purif. mAbBlue: clarified supernatant

Aggregates(early eluting in SEC)



SEC-Analysis of fractionation of preparative mAb gradient

elution: Aggregates are late eluting in CIEX.


0

2

4

6

8

10

12

80 85 90 95time [min]

conc

mA

b, M

onon

er [g

/L]

0

0.05

0.1

0.15

0.2

0.25

0.3

conc

Agg

[g/L

]

mAb conc [g/L]MonomerDimerTrimer


Monoclonal Antibody purification from cell culture supernatant:

Cell Culture supernatant is multi-component (fragments,

Aggregates, HCP, DNA)

Pure MAb is multi-component (variants)


MonoclonalAntibody (mAb):150 kDa

Purification of a mixture from a mixture


0

2000

4000

6000

8000

10000

12000

14000

16000

47 49 51 53 55 57 59 61

time [min]

conc

. [m

AU

s] (m

Ab)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

conc

. [m

AU

s] (

frag

men

ts),

[mA

U] (

onlin

e) mAb Propac

A280

W2

W3

0

2

4

6

8

10

12

80 85 90 95time [min]

conc

mA

b, M

onon

er [g

/L]

0

0.05

0.1

0.15

0.2

0.25

0.3

conc

Agg

[g/L

]

mAb conc [g/L]MonomerDimerTrimer

1. MAb purification challengeSummary (CIEX):

Three-fraction separation required to purify product.time

conc

. Strong(Aggregates)

Weak (Fragments + HCP)

Product


1. MAb purification challengeBatch chromatography:

If the desired purity is high, the achieved yield will be low!

Change the resin (e.g. use affinity chromatography)

Or …

Use a different process

time

conc

. Strong(Aggregates)

Weak (Fragments + HCP)

Product





4. Application examples



Batch versus continuous chromatography:- selective adsorption leads todifferent migration velocities

Features: Linear gradientsThree fraction separations

2. Continuous Liquid Chromatography

slow component

liquidflow

chromatographic column

fastcomponent


2. Continuous Liquid ChromatographyBatch versus continuous chromatography

liquidflow

slow solid flow


2. Continuous Liquid ChromatographyBatch versus continuous chromatography

liquidflow

fast solid flow


2. Continuous Liquid ChromatographyFrom batch to continuous countercurrent chromatography…

liquidflow

intermediate solid flow

?


• True Moving Bed • Design the unit with respect to an observer moving with the solid

2. Simulated Moving Bed Chromatography


2. Simulated Moving Bed Chromatography

22

SMB scheme:

Extract(strongly adsorbing)

Feed

Raffinate(early eluting) 44

11

33

Eluent


2. Batch versus Continuous ChromatographySeparation 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]








Batch chromatography: SMB:

pulsed feed

☺ multi-fraction separation☺ linear solvent gradients

low efficiency binary separationstep solvent gradients

☺ continuous feed☺ counter-current operation☺ high efficiency

3. Evolution of technologies

MCSGP (Multi-column Countercurrent Solvent Gradient Purification):


conc.weakstrong

Product

Elution time

3. MCSGP - Principle


3. Principle 6 Column Purification unit

all H outall P out

no H outall L out no P out

no P out


3. Principle 6 Column Purification unit

all H outall P out

no H outall L out no P out

no P out


all H out

all P out

no H out

all L out no P out

no P out


all H out

all P out

no H out

all L out no P out

no P out

3. Semicontinuous 3-Column Operation


3. Mobile MCSGP Unit

3 column MCSGP Process

- columns, multiposition

valves, gradient pumps

- UV/Cond./pH Monitor

- control computer

- based on Aekta/ Unicorn

- worldwide patent pending






5. MAb Variant Separation



70.0%

75.0%

80.0%

85.0%

90.0%

95.0%

100.0%

0 500 1000 1500 2000 2500HCP pool [ppm]

Yiel

d

Comparison of batch Protein A chromatography and

MCSGP (commercial HCP ELISA)

4. Results: MAb capture from cCCS

Protein A

MCSGP

Increasepurity


Comparison of batch Protein A chromatography and

MCSGP

MCSGP has ca. 10x higher HCP-clearance than Protein A

MCSGP reduces HCP by 2-3 logs


Conc. Purity Yield Prod.Pool Pool Pool

Mode Resin type SN dil cmab HCP[x-fold] [g/L] [ppm] [%] norm.

Batch Aff. Mab Select Sure PA 1 4.8 2036 82.0% 1*1st stepMCSGP Resin 1 run A SO3 4 2.7 146 94.9% 1.1MCSGP Resin 2 run B SO3 4 4.7 226 96.1% 4.8MCSGP Resin 2 run C SO3 3 4.9 625 96.0% 5.0

* Productivity of all runs normalized to Protein A run productivity


Excellent aggregate clearance

4. Results: Aggregate clearance

Aggregate content:Protein A: 0.8%MCSGP: 0.4%

Size exclusion chromatogram: Tosoh TSKgel G3000SWXL


Polishing CaptoAdhere (pH grad. 8.0-4.0) :

Purity of Pool: 99.5%, Yield 93.4 %

4. Results: Second purification step

0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

time [min]

conc

[g/L

], A

280

calib

rate

d

0102030405060708090100110120130140150160170180

cond

[mS/

cm],

pH*1

0 [-]

mAb Eluate

A280

imp

cond

pH

Capto Adhere


CIEX-MCSGP capture samples purified with Capto Adhere

4. Results: Full purification

Final product after 2-step process in specification (10 ppm)

Conc. Purity Yield Prod.Pool Pool Pool

Mode Resin type SN dil cmab HCP[x-fold] [g/L] [ppm] [%] norm.

Batch Aff. Mab Select Sure PA 1 4.8 2036 82.0% 1*1st stepMCSGP Resin 1 run A SO3 4 2.7 146 94.9% 1.1MCSGP Resin 2 run B SO3 4 4.7 226 96.1% 4.8MCSGP Resin 2 run C SO3 3 4.9 625 96.0% 5.02nd stepBatch Adhere polish A n.a. 3 2.0 1 96.1% 1.4Batch Adhere polish B n.a. 3 2.4 2 95.8% 2.3Batch Adhere polish C n.a. 3 2.2 3 94.3% 1.9* Productivity of all runs normalized to Protein A run productivity


Polishing Capto Adhere (complete removal of fragments):

4. Results: Full purification

Pink: cCCSBlue: MCSGP Red: MCSGP+Adh.Green: Final Product Serono

Analytical CIEX (Propac wCX-10, 4 x 250 mm)


0%10%20%30%40%50%60%70%80%90%

100%

95% 96% 97% 98% 99% 100%Purity

Yiel

d

Comparison of Pool fractions and purest fractions (Protein A

analysis):


Purest fractionin CIEX batch

CIEX batchchromato-graphy

CIEX MCSGP


3-step process replaced by 2-step process

5. Summary - MCSGP

Protein A

CIEX BE

AIEX FT

MCSGPCIEX

MMA BE


MCSGP: Internal recycling High yield and purity are achieved

simultaneously.

4. Results: MCSGP

0%10%20%30%40%50%60%70%80%90%

100%

95% 96% 97% 98% 99% 100%Purity

Yiel

d

Purest fractionin CIEX batch

CIEX batchchromato-graphy

CIEX MCSGP

time

conc

entra

tion

P

W SvW


4 columns required (3 columns perform purification, 1 is CIPped)

purificationCIP

4. MAb capture from cCCS (including CIP)

8 2 4 6

7 1 3 5

QCIP Q2, c2 Q4, c4 Q6, c6

Q1, c1 Q3, c3 QFeedQEquil

CIP S P W

DCIP

CCL:

BL:

8 2 4 6

7 1 3 5

QCIP Q2, c2 Q4, c4 Q6, c6

Q1, c1 Q3, c3 QFeedQEquil

CIP S P W

DCIP

CCL:

BL:


After 120 hrs of operation, headspace is visible in one column

(total cumulated operating time of columns up to that point: 400 hrs)

Pressure drop starts to increase


0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 2000 4000 6000 8000

time [min]

Pres

sure

dro

p [M

Pa] CIP no CIP


After 7200 min of operation, CIP with NaOH is re-established

Headspace disappears

Pressure drop reduced back to “normal”


0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 2000 4000 6000 8000 10000

time [min]

Pres

sure

dro

p [M

Pa] CIP no CIP CIP


CIP required for DNA clearance


0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

0 2000 4000 6000 8000 10000

time [min]

purit

y [n

g/m

g]

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

conc

entra

tion

[g/L

]

DNA/MAb [ng/mg]HCP/MAb [ng/mg]c Mab [g/L]c Mab (A280) [g/L]

CIP

no CIP CIP






5. Mab Variant Separation



Analytical Chromatogram on Propac WCX-10, pH 6.3, A220

I1 F1 F2 I2 F3

KK KKK

3 MAB variants with a variation in the constant part of the molecule

Characterization and design of preparative separation by ion-exchange

5. Antibody Variant Separation


5. Linear Gradient Elution

Purity of F2 < 80 %

ExperimentSimulation

Required:

PurityF2 > 80 %

YieldF2 > 90 %

Fractogel EMD COO 100x4.6 mm, dp = 30µm

Continuous Process


5. MCSGP – Purity and Yield of MAB Variant

Purity of F2 = 93%

Yield of F2 = 93%0

50

100

150

200

250

300

3 5 7 9time [min]

A 220

[mA

U]_

FeedF2 in P fraction

Purity of F2 in Fraction P

0%10%20%30%40%50%60%70%80%90%

100%

0 5 10 15 20 25time [h]

Purit

y of

F2

[-]

purity F2yield F2

Process Start-up


The ratio of the variants may be influenced by changing the

switch time tCC

Deamidatedvariants

Product variants

Protein A Pool contains all variants

5. Trends in mAb production


mAb product purity defined by … bacteria!

5. Trends in mAb production

Staphylococcus aureus






5. MAB Variant Separation

6. Comparison of Technologies


5. Comparison of technologiesProductivity as a function of mAb titer

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16

mAb titer [g/L]

prod

uctiv

ity [g

/L/m

in]

MCSGP 3x dilution

MCSGP no dilution

MCSGP 2x dilution

Protein A batchResults obtained from collaboration

Experimental Results:

• SN with 14 g/L mAb, no dilution

SN with 2.5 g/L, dil. 1:4

Prod

uctiv

ity[g

/L/h

]

mAb titer [g/L]


5. Trends in mAb productionAffinity and CIEX resin costs ($ per g mAb):

$0.0

$1.0

$2.0

$3.0

$4.0

$5.0

$6.0

$7.0

0 2 4 6 8 10 12 14 16

mAb titer [g/L]

resi

n co

sts

[US

$ / g

mAb

]

MCSGP 3x dilutionMCSGP 2x dilutionMCSGP no dilution

Protein A batch, $ 20000 / L

Protein A batch, $ 10000 / L


6. Summary

Continuous processes outperforms batch processes in terms of yield,

purity and productivity

All processes using Protein A can not relieve the cost pressure for

increasing mAb titers

MCSGP with ion-exchange reaches purity and yield comparable to

Protein A batch chromatography, and raises productivity

MCSGP can affect the MAB Safety and Potency


6. AcknowledgementsIndustrial Partners:

Merck Serono, Switzerland

Novartis, Switzerland

Lonza, UK

Chromacon AG, Switzerland

PhDs & Postdocs in preparative chromatography:Aumann, Dr. Lars Müller-Späth, Thomas Ströhlein, Dr. Guido