POROS® Affinity Chromatography Columns

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POROS® Affinity Chromatography Columns
PRODUCT BULLETIN
POROS® Affinity Chromatography Columns
POROS® Affinity Chromatography Columns
High-speed quantitation or purification
• Broad utility—POROS® A, G, and CaptureSelect® affinity
ligands address most biological
drug classes
• Rapid—POROS® resins offer fast quantitation or
purification of immunoglobulins
and fusion proteins
• Reduced hands-on time—HPLC
method can be automated for
streamlined operation
• Robust—deliver highperformance purification over hundreds of
chromatography cycles
Analytical groups supporting
biological drug development
efforts require rapid, sensitive,
and precise methods for
quantifying proteins from cell
cultures, fermentation harvests,
or purification intermediates. It
is critical that biopharmaceutical
manufacturers be able to monitor
bioreactor product titers to
determine when to harvest or
abort a reactor cycle.
POROS® Affinity Chromatography
Columns incorporate the
advantages of high-speed, high-performance analytical 20 µm POROS® chromatography
resins with the unique selectivity
of Protein A, Protein G, and
CaptureSelect® ligands (Figure
1). The combination allows
for 3-minute quantitation
of immunoglobulins, fusion
proteins, Fab fragments,
and bispecific antibodies.
Alternative assays lack the
combined advantages of speed,
selectivity, method automation,
and high precision. In addition,
the affinity-purified protein is
suitable for subsequent analysis
such as mass spectrometry.
VH
VL
CH1
CL
Protein L
CaptureSelect®
LC-Kappa or LC-Lambda
Protein A or G
CH2
CH3
CaptureSelect®
IgG Fc
Figure 1. POROS® A, G, and CaptureSelect®
ligands target multiple IgG epitopes.
This graphic shows the binding sites for
affinity ligands Protein A, Protein G, and
Protein L. Novel POROS® CaptureSelect®
ligands provide alternative specificities that
enable rapid binding for the quantitation or
purification of engineered immunoglobulins
(POROS® Affinity Chromatography Columns
represented in orange).
163
150
Absorbance (mAU)
The BAC and Life Technologies
collaboration creates a range
of ready-to-use HPLC columns
prepacked with CaptureSelect® affinity
ligands immobilized on POROS®
chromatography media. Designed for
the high flow rates necessary to provide
the real-time results demanded for
biopharmaceutical development and
quality assurance needs, the unique
selectivity of these columns will
support Process Analytical Technology
(PAT) for most biopharmaceutical
manufacturing process.
125
100
75
50
25
0
-20
0
•
•
•
•
•
•
IgG—all subclasses, specific for
human and many other species
(Table 1)
IgM—specific for human, mouse,
or rat
Kappa light chain—specific for
human, all subclasses
Lambda light chain—specific for
human, all subclasses
Human serum albumin
Fusion proteins, Fc, or human
serum albumin
1.0
1.5
2.0
Time (min)
Figure 2. POROS® CaptureSelect® resins exhibit high-speed and efficient chromatography,
regardless of the type of kappa light chain. This overlay of 5 kappa light chain runs on a
POROS® CaptureSelect® LC-Kappa column illustrates the speed and efficiency of the analysis.
Column: POROS® CaptureSelect® LC-Kappa column (Cat. No. 4469149, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200, flow rate
2 mL/min. Sample injection: 50 µL purified kappa light chain, 0.5 mg/mL. Equilibration buffer: phosphate buffer, 150 mM NaCl, pH 7.5.
Elution buffer: 12 mM HCl, 150 mM NaCl, pH 1.9. UV detection: 280 nm.
70
Absorbance (mAU•min)
High speed with high specificity
The combination of Protein A,
Protein G, and CaptureSelect® affinity
ligands combined with POROS® 20
µm resin provides a high–flow rate,
high-performance chromatography
solution for the rapid quantitation and
small-scale purification of:
0.5
60
50
40
30
20
10
0
POROS perfusion chromatography
allows the quantitation of product from
clarified harvest or partially purified
intermediates—typically in under
3 minutes. Moreover, only minimal
sample preparation (0.2 µm filtration)
is required, and the eluted product
is highly purified and suitable for
further analysis. Figure 2 illustrates
the chromatographic performance of
POROS® CaptureSelect® LC-Kappa
with five unique kappa Fab
fragments. The associated standard
curves, Figure 3, exhibit excellent
linearity. Additionally, Figure 4 displays
the specificity of the CaptureSelect®
HSA ligand.
®
0
10
20
30
40
50
60
70
80
90
Injection volume (μL)
Figure 3. Standard curves for POROS® CaptureSelect® LC-Kappa exhibit good linear range
(8x) and linear correlation coefficients (R2 range: 0.9991 to 1).
Column: POROS® CaptureSelect® LC-Kappa column (Cat. No. 4469149, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200, flow rate
2 mL/min. Sample injection: Specified volumes of purified kappa light chain, 0.5 mg/mL. Equilibration buffer: phosphate buffer, 150
mM NaCl, pH 7.5. Elution buffer: 12 mM HCl, 150 mM NaCl, pH 1.9. UV detection: 280 nm.
Table 1. Selection guide.
POROS® Protein A, G resins
Bimolecular type
POROS® CaptureSelect® resins
Protein A
Protein G
IgG-Fc
LC-Kappa
LC-Lambda
•
•
•
•
•
•
•
•
•
•
•
•
IgM
•
•
IgA, IgD, IgE
•
•
Fab, Fab´2
•
•
IgG1, IgG2, IgG4
IgG3
IgG, CH2 domain deleted
HSA
•
Human serum albumin
•
Albumin fusion protein
•
IgG Fc fusion protein
•
•
•
Specifications
Product
Protein-binding capacity (mg/mL)
POROS® CaptureSelect® IgG-FC
Species specificity
8.9
Human
POROS CaptureSelect LC-Kappa
14.5
Human
POROS CaptureSelect LC-Lambda
®
®
10.0
Human
POROS® CaptureSelect® HSA
2.6
Human
POROS CaptureSelect IgM
4.0
Human, mouse, rat
®
®
®
®
POROS Protein A
19.0
POROS® Protein G
9.0
®
Human, mouse, and 5 other species
Human, mouse, rat, and 8 other species
Materials
Component
Material of construction
Column
PEEK™ polymer or stainless steel
Frit
2 µm, titanium
POROS resin
®
CaptureSelect® ligand
Protein A, Protein G
Automation
POROS® Affinity Chromatography
Columns are suitable for use with
HPLC systems, which allow for the
automation of chromatography
methods, data collection, and data
analysis. In addition, the immobilized
affinity ligands enable one method
and buffer system to be used across
a class of molecules, streamlining
analytical approaches.
Crosslinked poly (styrenedivinylbenzene)
Single-domain monospecific antibody fragment
produced in Saccharomyces cerevisiae
Recombinant Protein A and Protein G
Multiple cycles
Proper sample preparation, adherence
to column-operating instructions,
and the use of an optimized columncleaning regime result in strong
reuse performance. POROS® Affinity
Chromatography Columns can be
cycled hundreds of times for costeffective and reproducible quantitation
or purification.
IgM
A
Absorbance (mAU)
Figure 4. POROS® CaptureSelect® HSA
resin exhibits high specificity for the
target molecule. The specificity of a
POROS® CaptureSelect® HSA column was
demonstrated by loading (A) 1 mg/mL
bovine serum albumin (BSA) and (B)
1 mg/mL monoclonal antibody samples. The
chromatograms show both BSA and antibody
eluting in the void volume, whereas HSA
fusion protein (C) elutes at approximately
2.9 min.
Column: POROS® CaptureSelect® HSA column (Cat. No.
4469151, 2.1 x 30 mm, PEEK™ polymer). System: Agilent 1200,
flow rate 1 mL/min. Sample injection: 100 µL purified HSA
fusion protein, 0.2 mg/mL. Equilibration buffer: phosphate
buffer, pH 7.0. Elution buffer: 0.5 M acetic acid, pH 2.5. UV
detection: 280 nm.
400
300
200
100
0
0
1
2
3
4
5
4
5
Time (min)
B
Absorbance (mAU)
500
400
300
200
100
0
0
1
2
3
Time (min)
C
Absorbance (mAU)
120
100
80
60
40
20
0
0
1
2
3
5
Time (min)
5
6
7
Speak to a technical specialist about how these columns can improve your current process, at [email protected],
or go to www.appliedbiosystems.com/poros/captureselect.
Ordering information
Product
Column dimension
Volume (mL)
Cat. No.
(diameter (mm) x length (mm))
POROS® Protein A
20 μm (stainless steel column)
2.1 x 30
0.1
1-5024-12
20 μm (PEEK polymer column)
2.1 x 30
0.1
2-1001-00
20 μm (PEEK™ polymer column)
4.6 x 50
0.8
1-5022-24
20 μm (PEEK polymer column)
4.6 x 100
1.7
1-5022-26
20 μm (PEEK polymer column)
10 x 100
7.9
1-5022-46
2.1 x 30
0.1
2-1002-00
20 μm (PEEK polymer column)
4.6 x 50
0.8
1-5122-24
20 μm (PEEK™ polymer column)
4.6 x 100
1.7
1-5122-26
20 μm (PEEK polymer column)
10 x 100
7.9
1-5122-46
2.1 x 30
0.1
4469151
20 μm (PEEK polymer column)
4.6 x 50
0.8
4469165
20 μm (PEEK™ polymer column)
4.6 x 100
1.7
4469170
20 μm (PEEK polymer column)
10 x 100
7.9
4469175
2.1 x 30
0.1
4469148
20 μm (PEEK polymer column)
4.6 x 50
0.8
4469153
20 μm (PEEK™ polymer column)
4.6 x 100
1.7
4469166
20 μm (PEEK polymer column)
10 x 100
7.9
4469171
20 μm (PEEK™ polymer column)
2.1 x 30
0.1
4469152
20 μm (PEEK polymer column)
4.6 x 50
0.8
4469164
20 μm (PEEK polymer column)
4.6 x 100
1.7
4469169
20 μm (PEEK™ polymer column)
10 x 100
7.9
4469174
20 μm (PEEK™ polymer column)
2.1 x 30
0.1
4469149
20 μm (PEEK polymer column)
4.6 x 50
0.8
4469162
20 μm (PEEK polymer column)
4.6 x 100
1.7
4469167
20 μm (PEEK™ polymer column)
10 x 100
7.9
4469172
20 μm (PEEK™ polymer column)
2.1 x 30
0.1
4469150
20 μm (PEEK™ polymer column)
4.6 x 50
0.8
4469163
20 μm (PEEK polymer column)
4.6 x 100
1.7
4469168
20 μm (PEEK™ polymer column)
10 x 100
7.9
4469173
™
™
™
POROS® Protein G
20 μm (PEEK™ polymer column)
™
™
POROS® CaptureSelect® HSA
20 μm (PEEK™ polymer column)
™
™
POROS® CaptureSelect® IgG-Fc
20 μm (PEEK™ polymer column)
™
™
POROS® CaptureSelect® IgM
™
™
POROS® CaptureSelect® LC-Kappa
™
™
POROS® CaptureSelect® LC-Lambda
™
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
CaptureSelect is a registered trademark of BAC BV. PEEK is a trademark of Victrex PLC. Printed in the USA. CO24355 0112
lifetechnologies.com

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