September 18th 2008www.morbidelli.ethz.ch
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
3September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
4September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
MAb obtained from Merck-Serono:
analyt. CIEX (Propac wCX):
1. MAb purification challenge
Batch pools:
Red: CIEXBlue: Protein A
mAb fragments, early eluting in CIEX
5September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
MAb obtained from Merck-Serono:
analyt. SEC (Tosoh):
1. MAb purification challenge
Red: Protein A purif. mAbBlue: clarified supernatant
Aggregates(early eluting in SEC)
6September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
MAb obtained from Merck-Serono:
SEC-Analysis of fractionation of preparative mAb gradient
elution: Aggregates are late eluting in CIEX.
1. MAb purification challenge
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
7September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
Monoclonal Antibody purification from cell culture supernatant:
Cell Culture supernatant is multi-component (fragments,
Aggregates, HCP, DNA)
Pure MAb is multi-component (variants)
1. MAb purification challenge
MonoclonalAntibody (mAb):150 kDa
Purification of a mixture from a mixture
8September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
9September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
10September 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. Application examples
5. Comparison of technologies
11September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
12September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
2. Continuous Liquid ChromatographyBatch versus continuous chromatography
liquidflow
slow solid flow
13September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
2. Continuous Liquid ChromatographyBatch versus continuous chromatography
liquidflow
fast solid flow
14September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
2. Continuous Liquid ChromatographyFrom batch to continuous countercurrent chromatography…
liquidflow
intermediate solid flow
?
15September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
• True Moving Bed • Design the unit with respect to an observer moving with the solid
2. Simulated Moving Bed Chromatography
16September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
2. Simulated Moving Bed Chromatography
22
SMB scheme:
Extract(strongly adsorbing)
Feed
Raffinate(early eluting) 44
11
33
Eluent
17September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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]
18September 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. Comparison of technologies
19September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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):
20September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
conc.weakstrong
Product
Elution time
3. MCSGP - Principle
21September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
3. Principle 6 Column Purification unit
all H outall P out
no H outall L out no P out
no P out
22September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
3. Principle 6 Column Purification unit
all H outall P out
no H outall L out no P out
no P out
23September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
all H out
all P out
no H out
all L out no P out
no P out
24September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
all H out
all P out
no H out
all L out no P out
no P out
3. Semicontinuous 3-Column Operation
25September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
26September 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
27September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
28September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
4. Results: MAb capture from cCCS
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
29September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
Excellent aggregate clearance
4. Results: Aggregate clearance
Aggregate content:Protein A: 0.8%MCSGP: 0.4%
Size exclusion chromatogram: Tosoh TSKgel G3000SWXL
30September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
31September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
32September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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)
33September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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):
4. Results: MAb capture from cCCS
Purest fractionin CIEX batch
CIEX batchchromato-graphy
CIEX MCSGP
34September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
3-step process replaced by 2-step process
5. Summary - MCSGP
Protein A
CIEX BE
AIEX FT
MCSGPCIEX
MMA BE
35September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
36September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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:
37September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
4. MAb capture from cCCS (including CIP)
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
38September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
After 7200 min of operation, CIP with NaOH is re-established
Headspace disappears
Pressure drop reduced back to “normal”
4. MAb capture from cCCS (including CIP)
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
39September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
CIP required for DNA clearance
4. MAb capture from cCCS (including CIP)
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
40September 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
41September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
42September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
5. Linear Gradient Elution
Purity of F2 < 80 %
ExperimentSimulation
Required:
PurityF2 > 80 %
YieldF2 > 90 %
Fractogel EMD COO 100x4.6 mm, dp = 30µm
Continuous Process
43September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
44September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
45September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
mAb product purity defined by … bacteria!
5. Trends in mAb production
Staphylococcus aureus
46September 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
47September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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]
48September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
49September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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
50September 18th 2008 Tecan Conference 2008 | Institute for Chemical and Bioengineering, ETH Zürich | Massimo Morbidelli
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