continuous chromatography for monoclonal antibody...
TRANSCRIPT
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
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2. Continuous Liquid ChromatographyBatch versus continuous chromatography
liquidflow
fast solid flow
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2. Continuous Liquid ChromatographyFrom batch to continuous countercurrent chromatography…
liquidflow
intermediate solid flow
?
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• True Moving Bed • Design the unit with respect to an observer moving with the solid
2. Simulated Moving Bed Chromatography
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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
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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