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

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© 2015 General Electric Company – All rights reserved.

First published January 2015

GE Healthcare Bio-Sciences AB, a General Electric Company.

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