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Imagination at work
New agarose based size exclusion chromatography (SEC) columns
GE Healthcare, Life SciencesJanuary, 2015
Content
1. Introduction
2. New GE SEC columns vs. predecessors
3. Application examples
4. Technical data
5. Summary
Download the latest edition of the
Size Exclusion Chromatography
Handbook
Introduction
3
Size exclusion chromatography (SEC) -basics
Separates molecules based on size
Characteristics
•Mild conditions
•Any buffer can
be used
•Non-binding
technique
•Limited in
sample volume
and speed
Well packed
columns
critical
Preparative runs
CiPP (Capture,
intermediate
Purification,
Polishing)
SEC -Polishing step for final high purity
Analytical runs
To determine purity
To calculate:% aggregates% fragments
4New agarose based SEC columns | Jan2015
Superdex™ 200 Increase and Superose™ 6 Increase columns…
…provide high resolution
separations under mild
conditions for proteins and other
biomolecules in the molecular
weight range Mr 10 000 to 600
000 and 5 000 to 5 000 000,
respectively.
The high-flow agarose based
medium tolerates harsh
cleaning and thus enables
consistent delivery of reliable
data.
New agarose based SEC columns | Jan2015
Pre-packed SEC columns from GE Healthcare
6
Re
so
lutio
n
Sample volumes
HiLoad™ Superdex
prep grade
columns
HiPrep™
Sephacry™l
columns
Superdex™/Superose™
µl ml
Range of sample volumes
Mr 102 103 104 105 106 107
Superdex Peptide
Superdex 75
Superdex 200 Increase
Superose 6 Increase
Superose 12
Superdex 200
Superose 6
Fractionation ranges Molecular weight ranges (globular
proteins) for each Superdex/Superose
media
Preparative
purification (up to 13
ml sample volume)
Small scale
reparative
purification and
analysis (up to
500 µl sample
volume)
In focus for this
presentation
New agarose based SEC columns | Jan2015
Replacing Superose™ 6 and Superdex™ 200 columns• Superose 6 and Superdex 200 columns have been on the market
since 1982 and 1992, respectively.
• Requirements on size exclusion chromatography analysis have
increased over the years and production techniques has evolved.
• GE Healthcare Life Sciences are now in the process of replacing
Superdex 200 and Superose 6 columns with a new platform. The new
columns are able to be used for the same applications as the
corresponding previous columns, since they have the same
fractionation range.
Superdex 200 columns – will be discontinued for sales Dec 31th 2015
Superose 6 columns – will be discontinued for sales Dec 31th 2016
7New agarose based SEC columns | Jan2015
Suitable applications and benefits
New SEC platform with increased
performance:
• Increased resolution
• Quicker runs
• Higher reproducibility
Benefits of pH-stable agarose
based SEC media:
• Prolong column life time
• Minimize carry-over between
runs
• Enables use of basic buffers
8
MAb’s and other antibodies
Membrane proteins
Fractionation range
10 kDa – 600 kDa
Protein complexes
Large proteins
Fractionation range
5 kDa – 5 000 kDa
*Adaptor-related protein complex 2, alpha 1
subunit; PDB rendering based on 1gw5.
From
http://en.wikipedia.org/wiki/Adaptor-
related_protein_complex_2,_alpha_1
Superdex™ 200
Increase
Superose™ 6
Increase
New agarose based SEC columns | Jan 2015
Three column sizes to fit different needs
Each column size is available for SuperdexTM 200 Increase and SuperoseTM 6
Increase
New agarose based SEC columns | Jan 2015 9
•High resolution analysis (25 – 500 µl sample volume)•Small-scale preparative runs (mg amount)
Tricorn™ 10/300 GL (10 mm x 300 mm glass column)
• Purity check
• Rapid screening
• Low sample (4 – 50 µl sample volume) and buffer consumption
Tricorn 5/150 GL (5 mm x 150 mm glass column)
• High resolution analysis (4 - 50 µl sample volume)
• Small-scale preparative runs (µg amount)
• Low sample and buffer consumption
Precision column 3.2/300
(3.2 mm x 300 mm glass column)
New size exclusionchromatography columns vs. predecessors
10
Superdex™ 200 Increase vs. Superdex 200
Increased resolution Quicker runs
New agarose based SEC columns | Jan 2015 11
Superdex 200 10/300 GL Superdex 200 10/300 GL Superdex 200 Increase
10/300 GL
Superdex 200 Increase
10/300 GL
0.5 ml/min -> 48 min1.5 ml/min -> 16 min
Up to 60% higher resolution
on Superdex 200 IncreaseUp to three times quicker runs
on Superdex 200 Increase with
retained resolution
0.5 ml/min -> 48 min
Sample mix of standard proteins were used in this comparison; Thyroglobulin (Mr 669 000), Ferritin Mr (440 000), Aldolase (Mr 158 000), Conalbumin (Mr 75 000),
Ovalbumin (Mr 43 000), Carbonic Anhydrase (Mr 29 000). Smallest proteins were different; for resolution comparison Aprotinin (Mr 6 500) was used and for Quicer
runs RNAse A (Mr 13 700) was used.
Superose™ 6 Increase vs. Superose 6
New agarose based SEC columns | Jan 2015 12
Superose 6 10/300 GL Superose 6 10/300 GL Superose 6 Increase 10/300
GL
Superose 6 Increase 10/300
GL
Increased resolution Quicker runs
0.5 ml/min -> 48 min1.0 ml/min -> 24 min
Up to 40% higher resolution
on Superose 6 Increase
Twice as fast with retained resolution
on Superose 6 Increase
0.5 ml/min -> 48 min
131205 Sup6Inc 10 300 10142694 PR1 B 001:10_UV1_280nm 131205 Sup6Inc 10 300 10142694 PR1 B 001:10_Inject
0
20
40
60
80
100
120
mAU
0.0 5.0 10.0 15.0 20.0 25.0 ml
131204 Sup 6 10161160 PR1 001:10_UV1_280nm 131204 Sup 6 10161160 PR1 001:10_Inject
0
20
40
60
80
100
120
mAU
0.0 5.0 10.0 15.0 20.0 25.0 ml
Sample mix of standard proteins were used in this comparison; Thyroglobulin (Mr 669 000), Ferritin Mr (440 000), Aldolase (Mr 158 000), Ovalbumin (Mr 43 000),
Ribonuclease A (Mr 13 700) was used.
Superdex™ 200 Increase and Superose™ 6 Increase complement each other
Superdex 200 Increase Superose 6 Increase
New agarose based SEC columns | Jan 2015 13
Sample: Mr
1. IgM 970 000
2. Thyroglobulin 669 000
3. Ferritin 440 000
4. BSA 66 000
5. Myoglobin 17 600
6. Vitamin B12 1 300
140324 10 300 10142694 LS585442 HMWIgM 001:10_UV1_280nm 140324 10 300 10142694 LS585442 HMWIgM 001:10_Inject
0
20
40
60
80
mAU
0.0 5.0 10.0 15.0 20.0 ml
1
2 3 4 56
Aggregates
140321 10 300 SDX200Inc 10136375 LS477302 HMWIgM 001:10_UV1_280nm 140321 10 300 SDX200Inc 10136375 LS477302 HMWIgM 001:10_Inject
0
20
40
60
80
mAU
0.0 5.0 10.0 15.0 20.0 25.0 ml
Aggregates + 1
2
3
4 56
Fractionation range: 5 – 5000 kDaFractionation range: 10 - 600 kDa
Superdex 200 Increase for MAb’s and other antibodies and Superose 6 Increase
for higher molecular weight proteins and protein complexes. Note. The broad no 1 peak for Superose 6 Increase was confirmed, by light scattering, to contain isoforms of IgM.
Highly reproducible results with Superdex™ 200 Increase 10/300 GL columns
New agarose based SEC columns | Jan 2015 14
20141016(1413466861)005:10_UV1_280nm 20141015(1413366777)005:10_UV1_280nm 20141015(1413383190)005:10_UV1_280nm
0
50
100
150
mAU
8.0 10.0 12.0 14.0 16.0 18.0 ml
Column-to-column Reproducibility
Overlay 3 different columns; Sample MAb5
Peak 1 MAb monomer (Mr150 000), Peak 2
FAb (Mr 50 000), Peak 3 DAb (Mr 13 000)
Resolution Retention volume
Results of comparison of 6 different Superdex 200 Increase media
batches:
Resolution – Minor differences, RSD < 6 %
Retention volume (ml) – Minor differences, RSD < 10 %
Batch-to-batch Reproducibility
1
2
3
The narrow specification for Superdex 200 Increase medium in combination with optimized column packing methods
results in high consistency and reproducibility both between columns as well as between different media batches.
Columns: Superdex 200 Increase 10/300
GL
Sample:
1. MAb5 (monomer) -Mr150 000
2. FAb (MAb5) – Mr 50 000
3. DAb (MAb5) - Mr 13 000
Sample volume: 50 µl (0.2% CV)
Buffer: 20 mM NaH2PO4 pH 7.4, 300 mM
NaCl
Flow rate: 0.75 ml/min
Detection: UV @ 280 nm
Application examples
Application examples
• Superdex™ 200 Increase:
• Three examples of MAb aggregate analysis
• Rapid detergent screening for membrane protein crystallization
• Superose™ 6 Increase:
• Oligomerization of a pathogenic protein
• Purification of a membrane protein complex
New agarose based SEC columns | Jan 2015
Superdex™ 200 Increase - MAb aggregate analysis 1 (3)
High resolution analysis of dimer and higher aggregates
1 2
3
0.0 5.0 10.0 15.0 20.0 25.0 30.0 Retention Time [min]
0
10000
20000
30000
Inte
nsi
ty [
µV
]
mIgG1 15 ug_0105_010 - CH2
1 2
3
0.0 5.0 10.0 15.0 20.0 25.0 30.0 Retention Time [min]
-2000
0
2000
4000
Inte
nsi
ty [
µV
]
mIgG1 15 ug_0105_010 - CH2
A. B.
Superdex 200 Increase 10/300 GL:
Analysis of monoclonal mouse IgG1
antibody aggregates
Column: Superdex 200 Increase 10/300
GL
Sample : Mouse IgG1, 1.0 mg/ml
Sample volume: 15 l
Flow rate: 0.6 ml/min
Buffer : 0.1 M Phosphate, 0.2 M NaCl, pH
6.8
System: HPLC system
New agarose based SEC columns | Jan 2015
Superdex™ 200 Increase - MAb aggregate analysis 2 (3)
Increased resolution (+50%) enables detection of MAb fragments
New agarose based SEC columns | Jan 2015 18
130301 10 300 10136375 Mab5 nr 2 001:10_UV1_280nm 130301 10 300 10136375 Mab5 nr 2 001:10_Inject
0
50
100
150
200
250
300
mAU
0.0 10.0 20.0 30.0 40.0 50.0 min
Rs 2.7
130301 10 300 10136375 Mab5 nr 2 001:10_UV1_280nm 130301 10 300 10136375 Mab5 nr 2 001:10_Inject
0.0
5.0
10.0
15.0
mAU
7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 ml
130306 SDX200 old 10111147 Mab5 001:10_UV1_280nm 130306 SDX200 old 10111147 Mab5 001:10_Inject
0.0
5.0
10.0
15.0
mAU
8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 ml
130306 SDX200 old 10111147 Mab5 001:10_UV1_280nm 130306 SDX200 old 10111147 Mab5 001:10_Inject
0
50
100
150
200
250
300
mAU
0.0 10.0 20.0 30.0 40.0 50.0 min
Rs 1.7
Sample: Monoclonal antibody,
Mab5 , conc 3.4 mg/ml
Sample volume: 100 µl
Flow rate: 0.5 ml/min
Buffer: PBS pH 7.4
Detection: UV @ 280 nm
System: ÄKTAmicro
Superdex 200 Increase 10/300 GL Superdex 200 10/300 GL
Superdex™ 200 Increase - MAb aggregate analysis 3 (3)
Quick detection of low level of aggregates
Superdex 200 Increase 5/150 GL:
Run time 4 min
Detection limit ≥0.1% aggregate for
this MAb.
New agarose based SEC columns | Jan 2015
Superdex™ 200 Increase – Rapid detergent screening for membrane protein crystallization
Down to 6 min analysis time by using short Superdex 200 Increase 5/150 GL column
New agarose based SEC columns | Jan 2015 20
Data courtesy of Dr Per Moberg, Karolinska Institute, Stockholm, Sweden
The recombinant membrane protein CE07 was solubilized in different detergents and column was equilibrated with buffer containing the same detergent. The
DDM run (chromatogram A) gave the most homogenous peak and was therefore considered the best choice for further crystallization experiments.
Superose™ 6 Increase - Oligomerization of a pathogenic protein
New agarose based SEC columns | Jan 2015 21
*Data courtesy of Dr. Joakim Bergström, Rudbeck Laboratory, Uppsala University
α-synuclein monomerA
Oligomerized
α-synuclein
Aldehyde
B
Superose 6 Increase 3.2/300 used for preparation
of oligomerized α-synuclein for further in-vitro and
in-vivo studies.
• Lewy bodies, abnormalities found inside nerve
cells in patients with Parkinson’s disease and
related neurodegenerative disorders, constitute
mainly aggregated forms of the protein α-
synuclein.
• Aldehydes, formed during oxidative stress, are
believed to be involved in the formation of Lewy
bodies.
Chromatogram A: Monomeric α-synuclein
Chromatogram B: After incubation with aldehyde,
monomeric α-synuclein has been oligomerized and
no monomeric form can be detected.
Superose™ 6 Increase - Purification of a membrane protein complex
New agarose based SEC columns | Jan 2015 22
GEHCtestSR6Inc005:10_UV1_280nm GEHCtestSR6Inc005:10_Inject
0
100
200
300
400
500
600
mAU
0.0 0.5 1.0 1.5 2.0 2.5 ml
SEC using Superose 6 Increase 5/150 GL of ATP synthetase complex
from E. coli membrane. Peak 1: Aggregates; Peak 2: Monomer protein
complex; Peak 3 and 4: Degradation products.
Peak 1
Peak 2:
ATP
synthetase
complex
Peak 3
Peak 4Superose 6 Increase 5/150 GL used as
second step for preparation of ATP
synthetase complex for use in
subsequent structural and molecular
mechanism studies.
Runtime: 16 min
• First purification step was affinity
chromatography using Ni
Sepharose™ Fast Flow
• In addition to small consumption of
sample and buffer, the separation
was achieved with low run time yet
high resolution.
Technical data
Superdex™ 200
Increase
Superdex 200 Superose™ 6
Increase
Superose 6
Matrix Cross-linked agarose and dextran Cross-linked agarose
Fractionation
range
Molecular weights from 10 000 to
600 000
Molecular weights from 5 000 to
5 000 000
Flow/pressure
properties
Rigid beads with
high flow/pressure
resistance
Regular Rigid beads with
high flow/pressure
resistance
Regular
Average bead size 8.6 µm 13 µm 8.6 µm 13 µm
pH stability
Regular use
Short term
(CIP)
pH 3 – 12
pH 1 - 14
Rigid, small beads with a narrow particle size distribution make the difference
New agarose based SEC columns | Jan 2015
Column Sample volume Max Flow rate Typical pressure
(over the packed
bed):
Column
dimensions/
column bed
volume
Superdex™ 200
Increase 10/300 GL
25 µl – 500 µl 1.80 ml/min 3.0 MPa 10 x 300 mm/24 ml
Superose™ 6
Increase 10/300 GL
25 µl – 500 µl 1.50 ml/min 3.0 MPa 10 x 300 mm/24 ml
Superdex 200
Increase 5/150 GL
4 µl – 50 µl 0.75 ml/min 3.0 MPa 5 x 150 mm/3 ml
Superose 6
Increase 5/150 GL
4 µl – 50 µl 0.75 ml/min 3.0 MPa 5 x 150 mm/3 ml
Superdex 200
Increase 3.2/300
4 µl – 50 µl 0.15 ml/min 3.0 MPa 3.2 x 300 mm/2.4
ml
Superose 6
Increase 3.2/300
4 µl – 50 µl 0.15 ml/min 3.0 MPa 3.2 x 300 mm/2.4
ml
Column characteristics – technical data
25New agarose based SEC columns | Jan 2015
Summary
Superdex™ 200 Increase and Superose™ 6 Increase columns…
…provide high resolution separations under mild
conditions for proteins and other biomolecules in the
molecular weight range Mr 10 000 to 600 000 and
5 000 to 5 000 000, respectively.
The high-flow agarose based medium tolerates
harsh cleaning and thus enables consistent delivery
of reliable data.
Get increased resolution and/or shorter runtime with higher reproducibility
New agarose based SEC columns | Jan 2015
Ordering information
Code no Product
28-9909-44 Superdex™ 200 Increase 10/300
GL
28-9909-45 Superdex 200 Increase 5/150 GL
28-9909-46 Superdex 200 Increase 3.2/300
29-0915-96 Superose™ 6 Increase 10/300 GL
29-0915-97 Superose 6 Increase 5/150 GL
29-0915-98 Superose 6 Increase 3.2/300
New agarose based SEC columns | Jan 2015 28
Download the latest edition
of the Size exclusion
chromatography Handbook
Like to know more about protein purification?
New agarose based SEC columns | Jan 2015 29
For further information about GE
solutions for protein purification, visit
http://www.gelifesciences.com/
For guidance on choosing the right
chromatography column, download
the Purify App
New agarose based SEC columns | Jan 2015 30
ÄKTA, Superdex, Superose and Tricorn are trademarks of GE Healthcare companies. GE, Imagination at work and GE Monogram
are trademarks of General Electric Company
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.
© 2015 General Electric Company – All rights reserved.
First published January 2015
GE Healthcare Bio-Sciences AB, a General Electric Company.
www.gelifesciences.com
GE Healthcare Bio-Sciences AB
Björkgatan 30
SE-751 84 Uppsala
Sweden
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