Data file 29-0670-18 AC

Ion exchange chromatography

Capto™ S ImpAct
Capto S ImpAct chromatography medium (resin) is a strong
cation exchanger (Fig 1). The medium is designed for polishing
of monoclonal antibodies (MAbs) and a wide range of other
biomolecules. Capto S ImpAct is part of a platform of media
based on the Capto product line. The polymeric ligand, in
combination with the attributes of the base matrix, gives a
cation exchanger with high binding capacity. By combining
the rigidity of Capto media with an optimized bead,
Capto S ImpAct delivers both good pressure/flow properties
and good resolution, even at high sample load. The high
binding capacity and resolution, combined with the ability to
run at high flow rates and bed heights, increase productivity
and flexibility in process design. Fig 1. Capto S ImpAct extends the Capto platform to include a high-binding
and high-resolution polishing chromatography medium.
Key benefits of Capto S ImpAct include:
• High binding capacity, typically > 100 mg MAb/mL medium
• Efficient aggregate removal at high load of MAbs Capto S ImpAct is based on a high-flow agarose base matrix
with good pressure/flow properties and an average bead
• High-resolution polishing
size of 50 μm, which combined with an optimized porosity
• Flexibility of design: large operational window of flow gives high resolution. These properties, in combination with
rates and bed heights for easy optimization and scaling the polymer ligand, make Capto S ImpAct a good choice for
• High productivity, enabling cost-efficient manufacturing reliable and robust polishing steps in an industrial purification
• Security of supply and comprehensive regulatory support process. Table 1 lists further characteristics of the medium.

Chromatography medium characteristics

Ionic groups and base matrix
Capto S ImpAct is a strong cation exchange (CIEX) medium.
The medium has a polymer-grafted ligand composed of a = Pyrrolidone
mix of two different building blocks, a negatively charged
sulfonate group and a neutral pyrrolidone (Fig 2). This = Sulfonate

composition combines a surface extender with functional

Fig 2. Schematic picture of the polymer-grafted Capto S ImpAct medium.
groups, which results in a high binding capacity. The polymer surface extender is formed by random grafting between the
two building blocks.

gelifesciences.com
Table 1. Main characteristics of Capto S ImpAct The static binding capacity (SBC) of Capto S ImpAct for three
MAbs and one polyclonal IgG (pIgG) was determined over a
Matrix High-flow agarose
wide range of conductivities and pH values using prefilled
Functional group SO3-
PreDictor™ Capto S ImpAct 96-well filter plates (Fig 3). The
Total ionic capacity 37 to 63 µmol (H+)/mL medium red areas in the contour plots in Figure 3 correspond to
Average particle size (d50v)1 50 μm the highest SBC (> 100 mg/mL) and the blue to the lowest.
Flow velocity2 Minimum 220 cm/h in a 1 m diameter The binding optimum differs between the antibodies, but
column with 20 cm bed height at 20°C; as indicated by the large red areas in all four contour plots,
measured using process buffers with
the same viscosity as water at < 3 bar Capto S ImpAct showed high SBC under a wide range of
(44 psi, 0.3 MPa) conditions for all antibodies tested.
Dynamic binding capacity3 > 100 mg IgG/mL medium
> 90 mg lysozyme/mL medium Medium: PreDictor Capto S ImpAct, 2 µL/well
> 85 mg BSA/mL medium Sample: ~ 3 mg/mL (A) MAb A, (B) MAb B, (C) MAb D, purified
on MabSelect SuRe™ chromatography medium, or (D)
pH stability (operational)4 4 to 12 commercial pIgG, buffer exchanged to 5 mM sodium
Cleaning-in-place stability 3 to 14 acetate, pH 5.4
(short term)5 Sample load: 200 µL
Working temperature 4°C to 30°C Start buffers: 50 mM sodium acetate, pH 4.5 to 6.0, and 0 to 350 mM
NaCl
Chemical stability All commonly used aqueous buffers, Incubation time:
> 100 mg/mL
90 minmg/mL
100–70 > 100 mg/mL
1 M sodium hydroxide (NaOH)6, 8 M urea, 70–40 mg/mL 100–70 mg/mL
6 M guanidine hydrochloride, 70–40 mg/mL
30% isopropanol, and 70% ethanol (A)
6.0 6.0
(B)
6.0
6.0 6.0
5.9 5.9 5.9
5.9 6.0 5.9
Avoid Oxidizing agents, cationic detergents 5.8 5.8 5.8
5.8 5.9 5.8
5.7 5.7 5.7
5.7 5.8 5.7
5.6 5.6 5.6
5.6 5.6
Storage 0.2 M sodium acetate in 20% ethanol at 5.5 5.5 5.5
5.5
5.7
5.6 5.5

pH 4.5–6.0

pH 4.5–6.0

pH 4.5–6.0
pH 4.5–6.0

pH 4.5–6.0
4°C to 30°C 5.4 5.4 5.4
5.4 5.5 5.4

pH 4.5–6.0
5.3 5.3 5.3
5.3 5.3
pH

pH
pH

pH
5.4
5.2 5.2 5.2
5.2 5.3 5.2

pH
5.1 5.1 5.1
5.1 5.2 5.1
1
d50v is the median particle size of the cumulative volume distribution. 5.0 5.0 5.0
5.0 5.0
5.1
4.9 4.9 4.9
4.9 5.0 4.9
2
Flow velocity stated in the table is dependent of the column used. 4.8 4.8 4.8
4.8 4.8
4.9
4.7 4.7 4.7
4.7 4.8 4.7
3
Dynamic binding capacity at 10% breakthrough measured at a residence time of 4.6 4.6 4.6
4.6 4.6
4.7
5.4 min in a Tricorn™ 5/50 column with 5 cm bed height (corresponding to 220 cm/h in 4.5 4.5 4.5
4.5 4.6 4.5
> 100 mg/mL
a 20 cm column). 50 mM sodium acetate, pH 5.5 (IgG), 25 mM sodium phosphate, pH 7.2 0 >50
50 0100 100 mg/mL
150100
200150250
200
300250 300 350
350 0 0 5050100
100 150200
150
4.5 200250
250300
300350
350 0
100–70 mg/mL 100–70 mg/mL
Concentration
Concentration (mM) (mM) 0 50(mM)
Concentration
Concentration 100 150 200 250 300 350
(mM)
(lysozyme), and 50 mM sodium acetate, pH 5.0 (BSA).
70–40 mg/mL 70–40mM
mg/mL
0–350 mM NaCl 0–350mM Concentration (mM)
mMNaCl
NaCl
0–350 NaCl 0–350
4
Working range: pH interval where the medium can be operated without significant 0–350 mM NaCl
change in function. (C) (D)
6.0 6.0 6.0 6.0 6.0
6.0
6.0 6.0 6.0
5
Cleaning in place: pH stability
5.9 where the medium can be subjected
5.9 to cleaning in place 5.9
5.9 5.9 5.9
5.9
5.9 5.9
5.8 5.8
without significant change
5.8 in function. 5.8 5.8
5.7
5.8 5.8 5.8
5.7
5.8
5.7 5.7 5.7 5.7 5.7 5.7 5.7
5.6 5.6
6
No significant change 5.6
in ionic capacity and carbon content after5.6storage for 1 week in 5.6
5.5
5.6 5.6 5.6
5.5
5.6
5.5 5.5 5.5 5.5
4.5–6.0
5.5 5.5 5.5
4.5–6.0

1 M NaOH at 40°C.
pH 4.5–6.0

pH 4.5–6.0

pH 4.5–6.0

5.4 pH4.5–6.0 5.4

pH4.5–6.0

pH 4.5–6.0

pH 4.5–6.0
5.4 5.4 5.4 5.4 5.4 5.4 5.4
5.3 pH 5.3
pH

5.3 5.3 5.3 5.3 5.3 5.3 5.3

pH

pH

pH

pH

5.2 5.2 5.2 5.2 5.2

5.2
5.2 5.2 5.2
5.1 5.1
pH
pH

5.1 5.1 5.1 5.1

High binding capacity
5.1 5.1 5.1
5.0 5.0 5.0 5.0 5.0
5.0
5.0 5.0 5.0
4.9 4.9 4.9 4.9 4.9
4.9
4.9 4.9 4.9
4.8 4.8 4.8 4.8 4.8
4.8
4.8 4.8 4.8
4.7 4.7
There is a growing need for MAb downstream processes that
4.7
4.6
4.7
4.6
4.7
4.6
4.6
4.7
4.6
4.7
4.6
4.7
4.6
4.6
4.7
4.6
4.5 4.5 4.5 4.5 4.5
4.5
4.5 4.5 4.5
can handle the challenges associated
0 50 100 150 200 250 300 350with increased
0 50 100 product
150 200 250 300 350 0 50
0 50 0 100
50 150
100 100 200
150 150 250
200 200 300
250 250 350
300 300 350
350
0 50 100
0 50 0 10050150
150
100200 200
150250 250
200300 300
300 350
250350 350 0 50 1
Concentration (mM) Concentration (mM)
titers upstream in a cost-effective
Concentration (mM)
0–350 mM NaCl
and efficient way. One way
Concentration (mM)
0–350 mM NaCl
Concentration
Concentration
0–350 mM
mM NaCl
0–350
0–350
(mM)(mM)
mM NaCl
NaCl
Concentration
Concentration (mM) (mM)
0–350 mM NaCl
0–350
0–350 mM mM NaCl
NaCl
Co

to improve productivity is to use chromatography media with

Fig 3. Contour
6.0 plots showing SBC of (A) MAb A;6.0
(B) MAb B; (C) MAb D; (D) pIgG
high dynamic binding capacities (DBC). Several studies using 5.9
in PreDictor
5.8
5.9
Capto S ImpAct 2 µL plates. Samples
5.8 were run in triplicate.
a selection of antibodies, listed in Table 2, have demonstrated 5.7
5.6
5.7
5.6
5.5 5.5
the high binding capacities of Capto S ImpAct (Fig 3 and 4).
pH 4.5–6.0
pH 4.5–6.0

5.4 5.4
5.3
pH

5.3
pH

The results show that Capto S ImpAct is a useful tool for these Robust dynamic binding capacity
5.2
5.1
5.2
5.1
challenges faced in today’s biopharmaceutical manufacturing. 5.0 5.0
The high DBC of Capto S ImpAct was demonstrated in a study
4.9
4.8
4.9
4.8
4.7 4.7
using a selection of MAbs and one pIgG (Fig 4). Residence
4.6 4.6
4.5 4.5
Table 2. Isoelectric point (pI) and aggregate content for MAbs used in the time on 0the50 column
100 150 200 was
250 3005.4
350 min in all experiments. The
0 50 100 150 200 250 300DBC
350
studies with Capto S ImpAct Concentration (mM) Concentration (mM)
at 10% breakthrough
0–350 mM NaCl was high (between 840–350 andmM122 mg/mL)
NaCl

MAb pI Aggregate content for the MAbs tested.

MAb A 8.9 7% Furthermore, a study was performed to investigate if DBC
MAb B 8.6 2% was affected by high MAb concentrations (Fig 5). The results
MAb C 8.9 to 9.0 2% show that the DBC is unaffected by sample concentration
between 5 and 25 mg MAb/mL for the tested MAb. The high
MAb D 8.5 4%
and robust DBC indicates that Capto S ImpAct is useful for
MAb E 7.3 to 7.5 2%
high-productivity processes.

2  29-0670-18 AC
 0
4 6 8
Residence time (min)

140

DBC 10% breakthrough (mg/mL)

120 Table 3. Results from the purification of four MAbs using Capto S ImpAct
100
MAb A MAb C MAb D MAb E
80

60
DBC (mg/mL medium) 108 92 118 122
40
250 Load (mg/mL medium) 80 64 80 85
20 4 Start aggregate content (%) 7 2 4 2
200
0
Aggregates at 90% monomer 0.9 0.6 1.2 0.6
MAb A

MAb B

MAb C

MAb D

MAb E

pIgG
recovery (%)
Velocity (cm/h)

150
8

Fig 4. DBC 100

of Capto S ImpAct for five MAbs and one pIgG. The samples, Start HCP
250 content (ppm) 34 1800 300 454
approximately 10 mg/mL medium16 of each five MAbs and pIgG were run in a HCP at 90% monomer recovery (ppm) 4 8 42 25 43
200
Tricorn 5/5050column at a flow corresponding to a residence time of 5.4 min.
The pH values of the start buffers were 5.5 for MAbs A, B, D, and pIgG, and Start protein A content (ppm) 4 1 1 <1

Velocity (cm/h)
150
5.25 for MAbs0 C and E. The start buffer for MAb A also contained 50 mM NaCl.
Protein A at 90% monomer
8 <1 <1 <1 <1
10 15 20 25 30 35 40
Bed height (cm) recovery100(ppm)
16
120 Elution pool volume (CV) 5.1 5.4 4.0 6.3
50
DBC 10% breakthrough (mg/mL)

100 Elution pool concentration at 90% 13.6 10.6 17.8 12.3

monomer0 recovery (mg/mL)
80 10 15 20 25 30 35 40
Bed height (cm)
60
120
40 Medium: Capto S ImpAct

DBC 10% breakthrough (mg/mL)

20 Column: 100
Tricorn 5/100, bed height 10 cm
Sample: 80 MAb E, purified on MabSelect SuRe medium
0
5 mg MAb B/mL 25 mg MAb B/mL Sample load:
60 85 mg/mL medium
Fig 5. DBC of Capto S ImpAct at different sample concentrations. The samples Start buffer: 50 mM sodium acetate, pH 5.3
40
(5 mg and3500
25 mg MAb B/mL in 50 mM sodium acetate, 70 10200
mM NaCl, pH 5.5) Elution buffer: 50 mM sodium acetate + 500 mM NaCl, pH 5.3
20
were run in a Tricorn 5/50 column at a flow corresponding
3000 60 175to a residence Flow rate: 0.35 mL/min, residence time 5.4 min
time of 5.4 min. Gradient: 0 Linear,
B/mL 0 to 350 mM NaCl in 20 CV
Conductivity (mS/cm)

150
2500 50 5 mg MAb 25 mg MAb B/mL
Aggregates (%)

125
2000 40
High resolution
mAU

100
1500 30
75 3500 70 200
A main MAb
1000 polishing purification challenge
20 is50 often to 3000 60 175

selectively
500 remove impurities similar to the target product.

Conductivity (mS/cm)
10 25 150
2500 50

Aggregates (%)
Examples0of impurities are protein fragments, 0 0 aggregated
2000 40
125
mAU

0 20 40 60 80 100 100
forms, and structural or mL charge variants, as well as protein A 1500 30
75
(from the initial capture step) and host cell proteins (HCP). 1000 20
50
Also, high antibody titers often increase the amount of these 500 10 25

impurities in the cell culture feedstock. Chromatography 0 0 0

0 20 40 60 80 100
media designed for polishing therefore need to offer high mL

resolution for effective removal of such impurities, while Fig 6. Chromatogram from purification of MAb E using Capto S ImpAct in a
retaining high recovery. The attributes of Capto S ImpAct Tricorn 5/100 column. Histogram in green represents aggregates in fractions.
offer an excellent solution for this challenge even at high Size exclusion chromatography was used for aggregate content analysis,
using a prepacked Superdex™ 200 Increase 10/300 GL column.
sample load.
To evaluate Capto S ImpAct for removal of aggregates, four
different MAbs purified on MabSelect SuRe medium were run High resolution for late-stage purification
using linear gradient elution in Tricorn columns. Fractions and polishing
from the elution peaks were collected and analyzed by GE Healthcare Life Sciences offer several strong cation
analytical gel filtration (size-exclusion chromatography) for exchange media. Even though the charged groups of the
aggregate content. HCP and protein A content was analyzed
sulfonate ligand are similar for these media, differences in
using a Gyrolab™ workstation and a commercial ELISA
base matrix, ligand density, and surface extenders give
assay, respectively. As can be seen in Table 3, Capto S ImpAct
differences in selectivity and resolution. Figure 7 presents
demonstrates effective aggregate and HCP removal at a
chromatograms from four ion exchange runs in prepacked
high monomer recovery. For example, MAb A, which had
HiScreen™ columns. As can be seen by the sharper peaks,
the higher initial aggregate content (7%) was nonetheless
Capto S ImpAct, Capto SP ImpRes, and SP Sepharose™
effectively purified from aggregates and other contaminants.
High Performance deliver improved resolution compared
Figure 6 shows the chromatogram for MAb E, illustrating that
with Capto S. The result is due to that the three former have
aggregates (green) elute at the tail of the elution peak. For
smaller average bead sizes, 50 µm, 40 µm, and 34 µm,
this MAb, the aggregate level was reduced to 0.6% at 90%
respectively, compared with Capto S (90 µm).
monomer recovery. The initial aggregate content was 2%.

29-0670-18 AC  3
 Column: (A) HiScreen Capto S ImpAct; Media: (A) Capto S ImpAct; (B) Capto SP ImpRes; (C) Eshmuno CPX;
(B) HiScreen Capto SP ImpRes; (C) HiScreen SP HP; (D) Fractogel SO3- (M); (E) Nuvia HR-S;
(D) HiScreen Capto S (F) Poros XS in 96-well plates, 2 µL medium/well
Sample: Protein mix of α-chymotrypsinogen A 4.5 mg/mL Sample: ~ 3 mg/mL MAb A, purified on MabSelect SuRe medium
Capto Smedium and lysozyme 3 mg/mL medium
Capto S ImpActin 20 mM Sample load: 200 µL
700 sodium phosphate, pH 6.8
1000 Start buffers: 50 mM sodium acetate, pH 4.5 to 6.0, and 0 to 350 mM NaCl
Sample
600 load: 3 mL
500
800 Incubation time: 90 min
Start buffer: 20 mM sodium phosphate, pH 6.8
mAU

mAU
400 600 > 100 mg/mL
Elution buffer: Start buffer + 500 mM NaCl 100–70 mg/mL
300
Flow rate: 0.9 mL/min (5.4 min residence
400 time) 70–40 mg/mL
200
Gradient: Linear, 0% to 100% elution
200 buffer in 20 CV (A) 6.0 (B) 6.0
100 5.9 5.9
System:
0 ÄKTA™ avant 25 0
6.05.8
5.95.7
6.0
5.9
5.8
5.7
6.0
5.9
0 25 50 75 100 125 150 0 25 50 75 100 125 150 5.85.6 5.8 5.6 5.8
mL mL 5.75.5 5.7 5.5 5.7

pH 4.5–6.0

pH 4.5–6.0
5.65.4 5.6 5.4 5.6
(A) (B) 5.55.3 5.5 5.3 5.5

pH

pH
SP Sepharose
Capto S HP Capto SP ImpRes
S ImpAct

pH 4.5–6.0

pH 4.5–6.0

pH 4.5–6.0
Capto S ImpAct 1000 5.45.2 5.4 5.2 5.4
700 5.35.1 5.3 5.3

pH

pH
1200 1000 5.1
1000 5.25.0 5.2 5.0 5.2
600 5.14.9 5.1 5.1
1000 800 4.9
800 800 5.04.8 5.0 4.8 5.0
500
4.94.7 4.9 4.7 4.9
800 600 4.84.6 4.8 4.8
4.6
mAU

mAU

400
mAU
mAU

mAU

600 600
4.74.5 4.7 4.5 4.7
600 4.6 0 50 100 150 200 250 300 350 4.6 4.6
300 0 50 100 150 200 250 300 350
400
400 4.5 4.5 4.5
400
400
200 Concentration
0 50 100 150 200 250(mM)
300 350 0 Concentration
50 100 150 200 250 300 (mM)
350 0 50 1
200 0–350 mM NaCl 0–350 mM NaCl
200
200
100
Concentration (mM) Concentration (mM) Co
0–350 mM NaCl 0–350 mM NaCl 0
000 00 (C) 6.0 (D) 6.0
25 150 00
0 25
25
25 50
50
50 75
75
75 100
100
100 125
125
125 150
150
150 00 25
25 50
50 75
75 100
100 125
125 150
150 5.9 5.9
mL
mL
mL mL
mL 5.8 5.8
5.7 5.7
(C) (D) 5.6 5.6
SP Sepharose
Capto HP
SP ImpRes Capto SP ImpRes
Capto S 5.5 Capto S ImpAct 5.5

pH 4.5–6.0
1000

pH 4.5–6.0
1000 700 5.4 5.4
1200
1000

pH
5.3 5.3

pH
800
1000
600
800 5.2 5.2
800 5.1 5.1
500 5.0 5.0
800 600 4.9 4.9
600
mAU

mAU

mAU

400
mAU

600 4.8 4.8

600 4.7 4.7
400 300
400 4.6 4.6
400
400 4.5 4.5
200
200 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
200
200 200
100 Concentration (mM) Concentration (mM)
00 0 0 0–350 mM NaCl 0–350 mM NaCl
25 150 00 25
25 50
50 75
75 100
100 125
125 150
150 0 25 50 75
75 100
100 125
125 150
150 0 25 50 75 100 125 150
mL
mL mL mL
(E) 6.0 (F) 6.0
5.9 5.9
Fig 7. Chromatograms showing different resolution, SP comparing
Sepharose HPfour cation
1000
5.8Capto SP ImpRes
5.7
5.8
5.7
ion exchange media. Peaks (left to right) are
1200
α-chymotrypsinogen A and 5.6 5.6
5.5 5.5
lysozyme. The chromatograms correspond to (A) Capto S ImpAct (50 μm);

pH 4.5–6.0
pH 4.5–6.0

1000 800
5.4 5.4
5.3

pH
5.3
(B) Capto SP ImpRes (40 μm); (C) SP Sepharose
800 High Performance (34 μm);
pH

600 5.2 5.2

mAU

mAU

5.1 5.1
(D) Capto S (90 μm). 600 5.0 5.0
400 4.9 4.9
400 4.8 4.8
4.7 4.7
200 4.6 4.6

Comparison to other media on the market

200
4.5 4.5
0
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350
0
0 25 50 75 100 125 150 0 25 50 Concentration
75 100 (mM) 150
125 Concentration (mM)
SBC and DBC studies as well as selectivity studies, show that mL mL
0–350 mM NaCl 0–350 mM NaCl

Capto S ImpAct has an improved performance compared with

five other commercially available cation exchange media*, Fig 8. Contour plots for determination of SBC of (A) Capto S ImpAct;
(B) Capto SP ImpRes; (C) Eshmuno CPX; (D) Fractogel SO3-(M); (E) Nuvia HR-S;
(Fig 8, 9, 10, and Table 4). The media tested were Capto S ImpAct,
(F) Poros XS for MAb A. Experiments were run in triplicate.
Capto SP ImpRes (GE Healthcare), Eshmuno™ CPX,
Fractogel™ EMD SO3- (M) (Merck Millipore), Nuvia™ HR-S
(Bio-Rad Laboratories), and Poros™ XS (Life Technologies). Based on SBC data, determination of DBC was performed
in triplicate runs at optimal conditions for each medium.
To find optimal running conditions for each medium, SBC
Figure 9 shows the DBC results for MAb A and MAb B,
for MAb A was measured over a wide range of pH and salt
demonstrating that Capto S ImpAct has higher DBC for
concentrations in a 96-well plate format. Samples were run in
the antibodies tested compared with the other cation
triplicate and the same conditions were used for all media.
exchangers included in the study.
Under the studied conditions, Capto S ImpAct has a SBC that
is higher or equivalent to the other media in the study (Fig 8).

* The comparison was performed in January and February, 2014, in Uppsala, Sweden.
The data is available at www.gelifesciences.com/captosimpactcomparison

4  29-0670-18 AC
Furthermore, Capto S ImpAct has a higher DBC, even at Media: Capto S ImpAct, Capto SP ImpRes, Eshmuno CPX, Fractogel
SO3- (M), Nuvia HR-S, or Poros XS
short residence times, compared with the other CIEX media
Column: Tricorn 5/50, 1 mL
tested (Fig 10).
Sample: 9.4 mg/mL MAb B, purified on MabSelect SuRe medium
Sample load: Until 10% breakthrough
Media: Capto S ImpAct, Capto SP ImpRes, Eshmuno CPX, Start buffer: 50 mM sodium acetate, pH 5.5
Fractogel EMD SO3- (M), Nuvia HR-S, or Poros XS
Elution buffer: Start buffer + 350 mM NaCl
Column: Tricorn 5/50, 1 mL
Flow rates: 0.12 to 0.24 mL/min, 4, 6, and 8 min residence time
Sample: MAb A and MAb B, purified on MabSelect SuRe medium
Sample load: Until 10% breakthrough
120
Start buffer: MAb A, pH 5.0: 50 mM sodium acetate, pH 5.0 + 50 mM NaCl Capto S ImpAct
(optimum for Capto S ImpAct, Eshmuno CPX, Fractogel Eshmuno CPX

DBC 10% breakthrough (mg/mL)

100
EMD SO3- (M), and Poros XS) Capto SP ImpRes
MAb A, pH 6.0: 50 mM sodium acetate, pH 6.0 (optimum 80
Poros XS
for Capto SP ImpRes and Nuvia HR-S) Fractogel EMD SO3- (M)
MAb B: 50 mM sodium acetate, pH 5.5 Nuvia HR-S
60
Elution buffer: Start buffer + 500 mM NaCl
40
Flow rate: 0.19 mL/min, 5.4 min residence time
20

120 0
MAb A, pH 5.0 4 6 8
100
MAb A, pH 6.0 Residence time (min)
MAb B
DBC 10% breakthrough (mg/mL)

140
Fig 10. Comparison of DBC at different residence times for Capto S ImpAct,

DBC 10% breakthrough (mg/mL)

80
120ImpRes, Eshmuno CPX, Fractogel EMD SO - (M), Nuvia HR-S, and Poros
Capto SP 3
60 XS. Experiments
100 were run in triplicate.
80
40
In addition
60 to the high SBC and DBC of Capto S ImpAct, the
20 selectivity
40
has also been shown to be good compared with
the other CIEX media studied, even at high sample load.
0 20
Capto S Capto SP Eshmuno CPX Fractogel Poros XS Nuvia HR-S Table 4 presents data for aggregate removal from MAb A for
ImpAct ImpRes EMD SO3- (M) 0
the different media. Samples were run in triplicate. The data
MAb A

MAb B

MAb C

MAb D

MAb E

pIgG
Fig 9. DBC comparison of different CIEX media. Capto S ImpAct exhibits equal shows that Capto S ImpAct has better selectivity for MAb A
or higher DBC compared with Eshmuno CPX. Capto SP ImpRes, Fractogel
than the majority of the tested media under the conditions
EMD SO3- (M), Nuvia HR-S, and Poros XS exhibit lower DBCs under the observed
conditions. Experiments were run in triplicate. studied.
In summary, the comparison studies show that Capto S ImpAct
is a competitive choice for high productivity processes.

Table 4. Aggregate removal data for Capto S ImpAct and four other commercially available CIEX media using MAb A

DBC Load 70% Recovery (%) at Load Recovery (%) at

(mg/mL of DBC (mg/mL 1% aggregate (mg/mL 1% aggregate
medium) medium) content STDEV* medium) content STDEV*
Capto S ImpAct 108 75 93 0.59 75 93 0.59
Eshmuno CPX 99 69 94 0.63 75 93 0.45
Fractogel EMD SO3 (M) -
73 51 85 1.85 75 Breakthrough
Nuvia HR-S 58 40 97 1.06 75 Breakthrough
Poros XS 65 45 91 1.29 75 Breakthrough
* STDEV = standard deviation

29-0670-18 AC  5
Reduced process costs
Higher titers in the upstream process often result in increased
challenges downstream, both in terms of higher mass and
more complex separation requirements. With a high DBC and
high resolution, enabling the use of smaller chromatography
medium volumes, column sizes, buffer volumes, and possibly
equipment footprint, Capto S ImpAct can be an efficient tool
to meet these challenges. The positive effects also stretch
beyond meeting the challenges in the downstream process.
Reducing the amount of chromatography medium required
greatly improves process economy in general.
When comparing Capto S ImpAct with Capto SP ImpRes
using BioSolve Process (BioPharm Services), the results show
that there is a significant improvement in process economy
for the CIEX step when using Capto S ImpAct compared with
Capto SP ImpRes. For this step in a standard MAb process,
using Capto S ImpAct could result in a 7% to 25% reduction in
total cost of goods/g MAb. In the simulation, the cost for labor
was set to identical for the two media and the cost for buffers
and water was seen as negligible. The CIEX step studied was
simulated in bind-and-elute mode.
Cleaning and sanitization
Cleaning in place (CIP) is a cleaning procedure to remove
impurities such as lipids, precipitates, or denatured proteins
that can remain in the packed column after regeneration.
Regular CIP also prevents the build-up of these impurities
in the chromatography bed and helps maintain the
capacity, flow properties, and general performance of the
medium. The frequency of CIP depends on the nature and
the condition of the starting material, but one CIP cycle is
generally recommended every one to five separation cycles.
Furthermore, a specific CIP protocol should be designed for
each process based on the type of impurities present.
100
90
DBC 10% breakthrough (mg/mL)
80
70
60
50
40
30
20
10
0 0
0 50 100 150 200 250 300
CIP cycle
0 15 40 45 60 75 90 105 120 135 150
Contact time (h)
Fig 11. Capto S ImpAct DBC is stable over 300 CIP cycles with 1 M NaOH.
Frontal analysis was performed at 4 min residence time on Tricorn 5/50 columns
packed with Capto S ImpAct. Sample concentration was 2.5 mg IgG/mL in
50 mM sodium acetate + 50 mM NaCl, pH 4.75. Each cycle comprised CIP
with 1 M NaOH for 30 min. Column 1 was only tested at start and end of the
study, whereas column 2 and 3 were tested at tighter intervals. Upper and
lower limits are the 95% confidence limits for the study.
6  29-0670-18 AC
The possibility of using NaOH in the CIP procedure is key in a
cost-effective production of pure MAb products at industrial
scale. In a life time study, Capto S ImpAct has shown great
alkaline stability. The results show that Capto S ImpAct
is stable over 300 purification cycles (Fig 11). Each cycle
comprised CIP with 1 M NaOH for 30 min.
Regular sanitization will prevent microbial growth and
maintain a high level of hygiene in the packed column.
A specific sanitization protocol should be developed based on
the nature and condition of the starting material.
Pressure/flow properties
Properties of Capto media, such as matrix rigidity, allow
a wide working range of flow velocities, bed heights, and
sample viscosities. High flow velocities increase volume
throughput and decrease process time. Higher bed heights
mean smaller diameters and a reduced column footprint.
Figure 12 shows the relationship between column bed height
and operating flow velocity for Capto S ImpAct. The size
of the area below the pressure-limit curve represents the
window of operation, that is, the available operating range
for the medium.
The pressure limits for Capto S ImpAct shown in Figure 12 are
based on a production-scale column and are calculated for
20 cm bed height and maximum operating flow velocities of
220 cm/h. At this flow velocity, the pressure is equal to or less
than 3 bar measured using process buffers with the same
viscosity as water, 20°C, which is the highest recommended
operating pressure for this medium at this scale. Capto S ImpAct
chromatography medium can normally be run at maximum
pressure ratings of low-pressure columns (3 bar).
Note that the maximum operating velocity of Capto S ImpAct
is dependent of the column used as well as the viscosity of
the liquid. For recommended columns, see Table 5.
500
Column 1
400
Column 2
Linear velocity (cm/h)
Column 3
Upper limit 300
Lower limit
4
200
8
100 16
0 10 20 30 40
Bed height (cm)
Fig 12. The Capto S ImpAct pressure/flow window of operation (area under
the blue curve). The window of operation describes schematically the limits
for the operating variables, here, the possible combinations of column bed
height and operating velocity. Data corresponds to a production-scale
column at 20°C and viscosity equivalent to water. Green lines show the
residence time in the column (4, 8, and 16 min).
Equipment Table 6. Main characteristics of HiTrap columns prepacked with Capto S ImpAct

Capto S ImpAct can be used with most modern chromatography Column volume 1 mL and 5 mL
equipment from laboratory to production scale. For details Column dimensions 0.7 × 2.5 cm (HiTrap 1 mL),
of packing laboratory-scale columns, see the appropriate 1.6 × 2.5 cm (HiTrap 5 mL)
Instructions for use. Table 5 lists suitable empty columns from Maximum flow rates 4 mL/min (HiTrap 1 mL),
GE Healthcare. Small-format columns prepacked with 20 mL/min (HiTrap 5 mL)
Capto S ImpAct are summarized in Tables 6 and 7. Recommended flow rates 1 mL/min (HiTrap 1 mL),
5 mL/min (HiTrap 5 mL)
Table 5. GE Healthcare column families suitable for packing Capto S ImpAct Maximum back pressure over 3 bar (44 psi, 0.3 MPa)
the packed bed
Column family Inner diameter (mm) Maximum pressure over the 5 bar (73 psi, 0.5 MPa)
Laboratory scale: hardware
Tricorn 5, 10
HiScale™ 16, 26, 50
Table 7. Main characteristics of HiScreen columns prepacked with Capto S ImpAct
Pilot and production scales:
AxiChrom™ 50 to 1000 Column volume 4.7 mL
BPG† 100 to 300 Column dimensions 0.77 × 10 cm
†
Note that the pressure rating of the BPG 450 column is too low for Capto S ImpAct medium. Maximum flow rates 300 cm/h (2.3 mL/min)
Recommended flow rates 100 to 300 cm/h

Small-scale formats give fast screening, method Maximum back pressure over 4 bar (58 psi, 0.4 MPa)
the packed bed
development, and small-scale purifications
Maximum pressure over the 8 bar (116 psi, 0.8 MPa)
Using small-scale formats for screening of the most suitable hardware
chromatography media and/or process conditions in the
early stages of process development saves both time and
sample. Capto S ImpAct is available in two sizes of prepacked Storage
96-well PreDictor plates (2 µL/well and 20 µL/well). Store unused Capto S ImpAct in 20% ethanol containing
The medium is also available together Capto SP ImpRes 0.2 M sodium acetate at 4°C to 30°C. Do not freeze the medium.
and Capto MMC ImpRes in PreDictor CIEX Capto polishing Store prepacked HiTrap Capto S ImpAct and HiScreen
screening plates as well as in PreDictor RoboColumn™ Capto S ImpAct columns in 20% ethanol with 0.2 M sodium
format. These miniaturized columns (200 µL and 600 µL sizes) acetate at 4°C to 30°C. Do not freeze the columns.
are prepacked with Capto S ImpAct for high-throughput After storage, equilibrate with suitable start buffer. An
process development (HTPD) using robotic liquid handling alternative storage solution is 2% benzyl alcohol containing
workstations. These products support HTPD by allowing 0.2 M sodium acetate.
parallel screening of chromatographic conditions such as pH
and conductivity.
BioProcess™ chromatography media
Capto S ImpAct is also available in the small prepacked
Capto S ImpAct is part of the BioProcess medium family
HiTrap™ 1 mL and 5 mL as well as HiScreen (4.7 mL column
comprising chromatography media widely used by
formats). Together with a chromatography system, such as
biopharmaceutical manufacturers. Support for these
ÄKTA pure or ÄKTA avant, prepacked HiTrap and HiScreen
products includes validated manufacturing methods, secure
columns are convenient to use when developing an efficient
long-term medium supply, safe and easy handling, and
and robust separation method. Basic characteristics of
regulatory support files (RSF) to assist process validation
HiTrap and HiScreen prepacked columns are summarized in
and submissions to regulatory authorities. In addition, the
Tables 6 and 7, respectively.
Fast Trak Training & Education team provides high-level
hands-on training for all key aspects of bioprocess
Scalability development and manufacturing.
Scale-up is typically performed by keeping bed height and
flow velocity constant, while increasing column bed diameter
and flow rate. However, as conditions are often optimized
using small column volumes, parameters such as DBC can be
optimized on shorter bed heights than those used in the final
scale. Nevertheless, as long as the residence time is constant,
the binding capacity for the target molecule remains the
same. Further development and optimization from, for
example, HiTrap and HiScreen prepacked columns are easily
done using HiScale columns.

29-0670-18 AC  7
Ordering information Related literature
Products Quantity Code number Data Files Code number
Capto S ImpAct 25 mL 17-3717-01 PreDictor 96-well filter plates and Assist software 28-9258-39
Capto S ImpAct 100 mL 17-3717-02 PreDictor RoboColumn 28-9886-34
Capto S ImpAct 1L 17-3717-03 HiScreen prepacked columns 28-9305-81
Capto S ImpAct 5L 17-3717-04 ReadyToProcess™ columns 28-9159-87
Capto S ImpAct 10 L 17-3717-05 AxiChrom columns 28-9290-41
Capto S ImpAct 60 L 17-3717-60 BPG columns 18-1115-23
HiTrap Capto S ImpAct 5 × 1 mL 17-3717-51
Application note
HiTrap Capto S ImpAct 5 × 5 mL 17-3717-55
Polishing of monoclonal antibodies using 29-1083-27
HiScreen Capto S ImpAct 1 × 4.7 mL 17-3717-47 Capto S ImpAct
PreDictor Capto S ImpAct, 2 µL 4 × 96-well filter 17-3717-16
Selection guides
plates
Comparison guide for process development tools 28-9951-64
PreDictor Capto S ImpAct, 20 µL 4 × 96-well filter 17-3717-17
plates Ion exchange columns and media 18-1127-31
PreDictor RoboColumn 8 columns in row 17-3717-71 Prepacked chromatography columns for ÄKTA systems 28-9317-78
Capto S ImpAct, 200 µL
Handbooks
PreDictor RoboColumn 8 columns in row 17-3717-72
Capto S ImpAct, 600 µL Ion Exchange Chromatography & Chromatofocusing: 11-0004-21
Principles and Methods
PreDictor Capto CIEX polishing 4 × 96-well filter 29-0955-68
screening, 2 µL/6 µL plates High-throughput Process Development with 28-9403-58
PreDictor plates: Principles and Methods
PreDictor Capto CIEX polishing 4 × 96-well filter 29-0955-67
screening, 20 µL plates Antibody Purification 18-1037-46

Related products
HiScale 16/20 1 28-9644-41
HiScale 16/40 1 28-9644-24
HiScale 26/20 1 28-9645-14
HiScale 26/40 1 28-9645-13
HiScale 50/20 1 28-9644-45
HiScale 50/40 1 28-9644-44

www.gelifesciences.com/CaptoSImpAct
GE and GE monogram and are trademarks of General Electric Company. ÄKTA, AxiChrom, BioProcess, Capto, HiScale, HiScreen, HiTrap,
MabSelect SuRe, PreDictor, ReadyToProcess, Sepharose, Superdex, and Tricorn are trademarks of General Electric Company or one of its
subsidiaries. Eshmuno and Fractogel are trademarks of Merck KGaA. Gyrolab is a trademark of Gyros Patent AB. Nuvia is a trademark of Bio-
Rad Laboratories Inc. Poros is a trademark of Life Technologies Corp. RoboColumn is a trademark of Atoll GmbH.
© 2014 General Electric Company—All rights reserved. First published Sep. 2014.
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them.
A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences AB GE Healthcare Europe, GmbH, Munzinger Strasse 5, D-79111 Freiburg, Germany
Björkgatan 30 GE Healthcare Bio-Sciences Corp., 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA
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751 84 Uppsala For local office contact information, visit www.gelifesciences.com/contact
Sweden 29-0670-18 AC 12/2014