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Gerd Kern,
October 16 - 18. 2012
Bioprocess Network Conference 2012 Melbourne
Increased productivity through constant chromatography innovations
2
Outline
1
2 Designing “state-of-the-art” downstream protein purification processes
Eshmuno® HCX – high salt-tolerant multi mode resin 3
4 ChromaSorb TM – anion exchange membrane adsorber
Eshmuno® CPX – cation exchange resin to increase monomer / aggregate separation efficiency 5
History and Industry challenges in bio-therapeutics manufacturing
The Need for Constant Innovation
New Challenges Demand Constant Innovation
Pressure on the Downstream Team
Cope with higher titers coming from
upstream processes
Increase process efficiency
Decrease time-to-market
Remain in compliance as
regulations evolve
Pressure on the Organization
Replenish pipelines
Compensate for the looming
patent cliff
Increase ROI on R&D
Streamline in the face of cost
pressures and decreasing
revenues
4
A Record of Innovation and Investment
First manufacturer of products
for chromatography
Starting one year after the
discovery of the chromatographic
principle in 1903
Ongoing long term
investment into R&D to
foster new Innovations
5
Recent Innovations
Eshmuno® HCX cation multi mode resin
For direct capture of recombinant proteins at higher salt
concentrations
ChromaSorb™ membrane adsorber portfolio
Single-use anion exchanger for impurity binding at
highest salt levels for MAb and protein purification
Eshmuno® CPX cation exchanger
For efficient monomer / aggregate separation
6
Expectations for Chromatography Media
Process challenges Desired media attributes
Increase throughput Flow rate
High Capacity
Increase purity Selectivity
Higher efficiency
At higher yield Capacity
Recovery
Reduce organic solvent
consumption
Shorter Cycle time
Live time
Reduce production cost
Chemical / mechanical stability
Sanitization stability (CIP)
7
Eshmuno® HCX Media
New, high salt-tolerant multi mode chromatography resin
9
Eshmuno® HCX Media Cation Exchanger for Direct Capture of Recombinant Proteins
The Eshmuno® HCX media is made
out of highly cross-linked polyvinyl
ether
Pressure flow properties to run up to
1000 cm / hr, 2,5 bar, 20 cm bed
height at large column id
Spherical regular shaped beads
with outstanding rigidity
Mean particle size 85 µm
Benefits
More selectivity
High binding capacity at high flow rate
Rigid base beads for easy and robust packing
Easy scalability
10
Eshmuno® HCX Media Flexible tentacle-type Ion Exchange for multi-point Interactions
Easy packing and scale-up of Eshmuno® HCX, similar like for the
other members of the Eshmuno® family
Flexible and easy accessible
ligands
Ionic group along the tentacle
polymer
Tentacles form a three-
dimensional ion exchange
network
20 years of experience
OH
O
NH
O
NH
O
NH
NH
O
NH
O
OHO
NH
SO
OO
O
O
O
O
O
O
SO
OO
O
O
O
O
SO
OO
weak ionic
strong ionic
hydrophobic
H-Bond
donor/acceptor
11
Eshmuno® HCX Media Binding Capacity – a Wide and Open Operation Window
pH 5.5 pH 6.0
150 mM NaCl 53 mg/ml 14 mg/ml
75 mM NaCl 27 mg/ml n.d.
Static binding capacity in
mg/ml IgG in 25 mM acetate,
25 mM phosphate,
0 – 300 mM NaCl
Dynamic binding capacity of
IgG in 20 mM phosphate, at
10% breakthrough, residence
time 5 min
pH
pH
7.0 6.5 6.0 5.5 5.0 4.5 4.0
300 mM NaCl
150 mM NaCl
75 mM NaCl
0 mM NaCl
60-80 40-60 20-40 0-20
Eshmuno® HCX Media Placing within purification train
The Multi-Mode Cation Exchange Media is specifically designed for the
direct capture of proteins at higher salt concentrations
CLARIFICATION
Millistak+®
Pod
CEX CHROMA-
TOGRAPHY
Eshmuno® HCX
AEX CHROMA-
TOGRAPHY
ChromaSorbTM
Eshmuno® Q
Fractogel® TMAE
FINAL FILL AND FINISH
UPSTREAM PROCESSING
Bioreactor
12
Binding and gradient elution of mAb2 from CCS acidified to pH 6.0 on Eshmuno® HCX
The mAb concentrations calculated from SEC data are shown in A for selected fractions. The doted line marks the 5% mAb break-
through. HCP contents in elution fractions were determined by ELISA. CSS, flow-through, and mAb02 elution fractions are analyzed with
Coomassie Blue stained SDS-PAGE under non-reducing conditions.
13
total load at 17 mS/cm 147 mg mAb2
150 kDa
UV 280 nm
Conductivity
0
500
1000
1500
2000
2500
mAU
0 50 100 150 200 250 300 350 ml
F2 A2 A4 A6 A8 A10 A12 B2 B4 B6 B8 B10 B12 C2 C4 C6 C8 C10 D1 D3 D5 D7 D9 D11 E1 E3 E5 E7 E9 E11 F1 F3 F5
CCS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
mAb (mg/ml)
flow - through 6 mg mAb2
elution pool 114 mg mAb2
B
0
50000
100000
150000
200000
250000
300000
HCP (ng/mg mAb )
total load at 17 mS/cm 147 mg mAb2
150 kDa
UV 280 nm
Conductivity
0
500
1000
1500
2000
2500
mAU
0 50 100 150 200 250 300 350 ml
F2 A2 A4 A6 A8 A10 A12 B2 B4 B6 B8 B10 B12 C2 C4 C6 C8 C10 D1 D3 D5 D7 D9 D11 E1 E3 E5 E7 E9 E11 F1 F3 F5
CCS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
mAb (mg/ml)
flow - through 6 mg mAb2
elution pool 114 mg mAb2
0
50000
100000
150000
200000
250000
300000
HCP (ng/mg mAb )
A
B
Binding and gradient elution of mAb2 from CCS acidified to pH 6.0 on Eshmuno® HCX
Minutes
0 5 10 15 20
co
unts
0
500000
1,0e+06
1,5e+06
co
unts
0
500000
1,0e+06
1,5e+06
8,9
37
Minutes
0 5 10 15 20
co
unts
0
100000
200000
300000
co
unts
0
100000
200000
300000
8,9
13
Minutes
0 5 10 15 20
co
unts
0
250000
500000
750000
co
unts
0
250000
500000
750000
8,9
23
CCS
flow-
through
and
wash
eluted
protein
C
D
E
mAb02
Minutes
0 5 10 15 20
co
unts
0
500000
1,0e+06
1,5e+06
co
unts
0
500000
1,0e+06
1,5e+06
8,9
37
Minutes
0 5 10 15 20
co
unts
0
100000
200000
300000
co
unts
0
100000
200000
300000
8,9
13
Minutes
0 5 10 15 20
co
unts
0
250000
500000
750000
co
unts
0
250000
500000
750000
8,9
23
CCS
flow-
through
and
wash
eluted
protein
C
D
E
mAb02
SDS-Page and SEC show significant
reduction of by-products.
HCP content in the feedstream is
reduced by a factor of at least 6 in the
elution pool.
The binding capacity was 29 mg mAb02
/ ml resin and the recovery was >80%.
14
15
7.06.56.05.55.04.5
300mM NaCl
150mM NaCl
75 mM NaCl
0 M NaCl
pH
80-100
60-80
40-60
20-40
0-20
7.06.56.05.55.04.5
300mM NaCl
150mM NaCl
75 mM NaCl
0 M NaCl
pH
80-100
60-80
40-60
20-40
0-20
7.06.56.05.55.04.5
300mM NaCl
150mM NaCl
75 mM NaCl
0 M NaCl
pH
80-100
60-80
40-60
20-40
0-20
7.06.56.05.55.04.5
300mM NaCl
150mM NaCl
75 mM NaCl
0 M NaCl
pH
80-100
60-80
40-60
20-40
0-20
Eshmuno® HCX Media SBC in comparison to other resins
Eshmuno® HCX
Eshmuno® S
Fractogel® SO3
Mixed-Mode
Static binding capacity of IgG in mg/ml settled resin measured in 25 mM acetate/25 mM phosphate pH 4.5- 7.0 with 0 - 300 mM NaCl in micro-titer plates
ChromaSorb™ Membrane Adsorber
Single-use anion exchanger for impurity binding at highest salt
levels for MAb and protein purification
ChromaSorbTM Membrane Adsorber
Single-use, flow-through anion exchange membrane
Designed to remove trace impurities including HCP, DNA, endotoxins,
and viruses as an anion column replacement in a mAb process
Robust impurity removal over a wide conductivity range
The result: significantly reduces buffer volumes, validation
requirements, and capital equipment expenditures
17
Benefits
Greater capacity at high salt concentrations
Linearly scalable family of devices
Single-use device provides enhanced
process flexibility and efficiency
Smaller manufacturing footprint than traditional
anion resins and columns
Eshmuno® CPX – a new media to come soon
New resin for efficient monomer / aggregate separation
Eshmuno® CPX Resin
Strong cation exchange resin build on proven Eshmuno® resin
technology.
Combines high aggregate removal efficiency in downstream
purification linked to an outstanding high dynamic binding capacity
The result: High resolution to meet the needs for highly productive
downstream purification bioprocessing applications.
21
Benefits
Excellent mAb monomer / aggregate separation efficiency
High resolution intermediate purification
High dynamic binding capacity
Excellent pressure flow behavior
Optimized resin characteristics:
Eshmuno® CPX media
22
• strong cation exchange resin
• 50 µm base bead
• optimized pore system
→ excellent mAb monomer/aggregate
separation efficiency
25
25
25
25
25
75
75
75
75
75
125
125
125
125
0
0
0
0
50
50
50
50
50
100
100
100
100
100
4,5 5,0 5,5 6,0
4
6
8
10
12
14
16
0
50
100
150
DBC pIgG [mg/mL CV]
pH
Co
nd
uc
tivit
y [
mS
/cm
]
Eshmuno® CPX media: Dynamic pIgG binding capacity*
res. time = 4 min
column vol. = 3.14 mL
elution buffer:
20mM sodium acetate +
20mM sodium phosphate +
1M sodium chloride
→ high dynamic binding capacities at
a large window of operation. * in mg/mL CV
pH
Co
nd
ucti
vit
y [
mS
/cm
]
90
90
90
90
90
90
70
70
80
80
80
80
80
80
80
60
4,00 4,25 4,50 4,75 5,00 5,25
5
6
7
8
9
10
11
12
pH
70
70
70
70
90
90
90
90
90
90
70
70
70
50
50
5030
80
80
80
80
80
80
80
80
100100
60
60
6040
20
4,00 4,25 4,50 4,75 5,00 5,25
5
6
7
8
9
10
11
12
Competitor 1 (Polymer) Competitor 2 (Agarose)
Eshmuno® CPX media: mAb SBC plots*
Eshmuno® CPX media shows highest mAb binding capacities
* SBC in mg/mL settled resin
pH
Co
nd
ucti
vit
y [
mS
/cm
]
110
110
130
130
130
130130
130
130
130130
110
110
90
90
70
50
120
120
120
120
140
140
140
140
120
120
120
100
100
80
60
40
4,00 4,25 4,50 4,75 5,00 5,25
5
6
7
8
9
10
11
12
Eshmuno® CPX resin
Eshmuno® CPX media: Superior mAb binding capacity Dynamic mAb binding capacities*
pH 4.75, 8 mS/cm
pH 5.75, 5 mS/cm
* in mg/ml CV
res. time: 5 min
1 ml scout column
compression: 15 %
65
99
0 20 40 60 80 100
1
2
DBC5% [mg/ml]
Eshmuno® CPX
Competitor (agarose)
52
80
0 20 40 60 80 100
1
2
DBC5% [mg/ml]
Eshmuno® CPX
Competitor (agarose)
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 20 40 60 80 100
ag
gre
ga
tele
ve
l [%
]
mAb monomer yield [%]
Eshmuno CPX - Load: 70 mg/ml
Competitor (agarose) - Load: 50 mg/ml
initial aggregate level: 7.2%
Excellent monomer/aggregate separation
mAb feedstream 1
Feed: mAb05 (post-ProtA pool), aggregate level 7.2 %
Column size: 5 mm i.d. x 200 mm; Column vol. : 3.9 ml
Linear flow rate: 250 cm/h
Buffer A: 25mM sodium acetate + 50mM sodium chloride,
pH 4.75, 8 mS/cm
Buffer B: 25mM sodium acetate + 1M sodium chloride,
pH 4.75
Gradient: 0 to 50 %B, 20 CV
Load: 70 mg/mL
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0 20 40 60 80 100
ag
gre
ga
te le
ve
l[%
]
mAb monomer yield [%]
initial aggregate level: 8.2%
Excellent monomer/aggregate separation
mAb feedstream 2
Feed: mAb07 (post-ProtA pool), aggregate level 8.2%
Column size: 5 mm i.d. x 200 mm; Column vol. : 3.9 mL
Linear flow rate: 250 cm/h
Buffer A: 50mM sodium acetate + 17mM sodium chloride,
pH 5.5, 5 mS/cm
Buffer B: 50mM sodium acetate + 1M sodium chloride,
pH 5.5
Gradient: 0 to 50 %B, 20 CV
Load: 50 mg/mL
HCP + leached protA clearance
0.0%
0.5%
1.0%
1.5%
2.0%
40 50 60 70 80 90 100 ag
gre
gate
lev
el
[%]
mAb monomer yield [%]
initial aggregate level: 2.9 %
Feed: mAb06 (post-ProtA pool), aggregate level 2.9 %
Column size: 8 mm i.d. x 100 mm; Column vol. : 5.0 ml
Linear flow rate: 250 cm/h
Buffer A: 25mM sodium acetate + 50mM sodium chloride,
pH 4.75, 8 mS/cm
Buffer B: 25mM sodium acetate + 1M sodium chloride,
pH 4.75
Gradient: 0 to 50 %B, 20 CV
Load: 50 mg/mL
HCP and leached protA clearance:
Load
(post-protA pool)
CPX
elution pool*
HCP level
[ng/mg mAb] 350 5
protA level
[ng/mg mAb] 3 <0.06
*Pooling criterion: aggregate level ≤ 0.5%
mAb feedstream 3
Eshmuno® CPX media: High resolution even at high flow rates
0
50
100
150
mAU
0.0
20.0
40.0
60.0
80.0
mS/cm
0 50 100 150 200 250 300 ml
200
78 cm/h
200 cm/h
600 cm/h
Separation of chymotrypsinogene A, cytochrome C and lysozyme;
Buffer A: 20 mM sodium phosphate, pH 6.0; Buffer B: 20 mM sodium phosphate + 1 M sodium chloride, pH 6.0
Eshmuno® CPX media: High resolution at high protein load
0
500
1000
1500
2000
2500
mAU
0
50
100
150
mS/cm
0 50 100 150 200 250 300 ml 350
60% loading*, 150 cm/h
60% loading*, 300 cm/h
60% loading*, 600 cm/h
Separation of chymotrypsinogene A and lysozyme;
Buffer A: 20 mM sodium phosphate, pH 6.0; Buffer B: 20 mM sodium phosphate + 1 M sodium chloride, pH 6.0
Alkaline stability of Eshmuno® CPX media
Run 1_UV_295nm
Conductivity
Run 50_UV_295nm
Run 101_UV_295nm
0
200
400
600
800
1000
mAU
0
50
100
150
mS/cm
0.0 10.0 20.0 30.0 40.0 min
Separation of chymotrypsinogen A, cytochrome C and lysozyme on Eshmuno® CPX,
overlaid chromatograms of run 1 (blue) run 50 (red) and run 101 (green).
•100 cycle study
•Cleaning of column after each
run with 2 CV 1 M NaOH
•Cleaning time 60 minutes
•Flow rate 20 cm/hr
Eshmuno® CPX media: Stability against various chemicals
5 M Urea
Separation of a mixture of Chymotrypsinogen A, Cytochrom C and Lysozyme
before and after treatment with
0
50
100
150
mAU
0 100 200 300 ml
6 M Guanidine·HCl 30% Isopropanol
0 100 200 300 ml 0
100 200 300 ml
Separation of chymotrypsinogene A, cytochrome C and lysozyme;
Buffer A: 20 mM sodium phosphate, pH 6.0; Buffer B: 20 mM sodium phosphate + 1 M sodium chloride, pH 6.0
Virus Removal Capabilities
These data demonstrate that under appropriate operating conditions, Eshmuno® CPX
media showed >4.0 logs clearance for retrovirus. By contrast, removal of parvovirus MVM
was less effective with less than 1 log removal.
Type of virus Total virus hold
(log10TCID50)
Total virus eluted
(log10TCID50)
LRV
XMuLV (retrovirus) 6.47 ≤ 2.41 ≥ 4.1
MVM (parvovirus) 7.28 6.91 0.4
34
Conclusions
Eshmuno® HCX media, our newest member of the Eshmuno® family, demonstrating a
unique performance for the capture of proteins at higher salt concentrations.
Eshmuno® HCX media is a cation exchange resin that maintains a high binding capacity
over a wide range of pH and salt concentrations, enabling users to operate at different
process conditions without the need for dilution.
ChromaSorbTM is a strong anion membrane adsorber that has been optimized for
removing negatively charged impurities at high salt concentrations in a flow through
mode.
Eshmuno® CPX media uses optimized resin properties combined with strong cation
chemistry to achieve an excellent mAb monomer / aggregate separation efficiency.
Merck Millipore is committed to long-term development investment for new
chromatographic innovations to address future industry challenges.
Thank You
35
Acknowlegements:
Merck Millipore Chrom R&D
Matthias Joehnck, Lars Peeck
