The Challenge:
Are you working with a sample that shows a tendency to
stick to traditional size exclusion resins with delayed elution
time, low recovery, varying HMWS, or excessive tailing?
Sepax SEC C-line columns!
Some comparison examples on the two different surface chemistries on size
exclusion separation
•
•
•
•
ADC
Fusion Protein
mPEG-peptide
Hydrophobic Peptide
•
•
•
•
MAb
Glycoprotein
PEGlylated peptide
Membrane Protein
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SEC phases
Difference in Phase Structures
Tough samples:
ADC, Derivatized MAb,
Pegylated Protein,
Membrane Protein,
“Super sticky protein”,
and etc.
Characteristics
Zenix, SRT
Zenix-C, SRT-C
Particle size
3 mm, 5 mm
Zenix 80, 100, 150, 300,
SRT 100, 150, 300, 500, 1000 & 2000
Chemically bonded stand-up monolayer
3 mm, 5 mm
Zenix-C 80,100, 150, 300
SRT-C 100, 150, 300, 500, 1000 & 2000
Chemically bonded lay-down monlayer
Versatile for mAbs, proteins, peptides,
oligonucleotides, virus, VLPs, and watersoluble polymers
Ideal for hydrophobic samples such as insulin or
ADC, hydrophobic mAb, pegylated peptides and
proteins
Pore size (Å)
Surface structure
Applications
SRT SEC-10 (10 mm) is the latest addition for semi-prep and prep scale separation.
SRT SEC-1000 and 2000 are suitable for very large biomolecule separation.
2
Herceptin Analysis on ZenixTM SEC-300 ( 7830 )
Column: ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM phosphate buffer
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin 2.34 mg/mL
mAU
70
60
Rs=1.93
50
40
30
20
10
0
2
4
6
8
3
10
12
min
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Herceptin Lysine ADC Analysis on SEC-300 ( 7830 )
Column: ZenixTM SEC-300, ZenixTM - C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM phosphate buffer
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin-lysine conjugate 2.05 mg/mL
mAU
100
80
60
40
20
0
ZenixTM - C SEC-300
ZenixTM SEC-300
2
4
6
8
4
10
12
14
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Herceptin-Lysine Drug Conjugate Analysis
Zenix-C SEC-300 vs. Tosoh TSKgel G3000SWXL
Column: Zenix® - C SEC-300 (3 µm, 300 Å, 7.8 x 300 mm) , Tosoh TSKgel G3000SW XL(5 µm, 250 Å, 7.8 x 300 mm)
Mobile phase: 150 mM phosphate buffer, pH 7.0; Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin-lysine drug conjugate 2.05 mg/mL
mAU
100
Rs=1.45, total area 2370
80
60
Rs=1.18, Total area 2273
40
20
0
Zenix-C
Tosoh TSKgel G3000SWXL
4
6
8
10
12
14
min
Disclaimer: TSKgel and Tosoh Bioscience are registered trademarks of Tosoh Corporation; Comparative
separations may not be representative of all applications.
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Herceptin Lysine ADC Analysis on SEC-300 ( 7830 )
Column: ZenixTM - C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: as indicated
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin lysine conjugate 2.05 mg/mL
mAU
1. 150 mM phosphate, pH 7.0
Rs=1.41, tailing=1.68
2. Buffer 1: 100% IPA = 95%:5%
Rs=1.50, tailing= 1.44
3. Buffer 1:100% IPA = 90%:10%
Rs=1.45, tailing= 1.35
25
20
mAU
120
100
80
60
40
20
0
15
4
6
8
10
12
min
10
5
0
2
4
6
8
6
10
12
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Herceptin Cysteine ADC (cleavable linker) Analysis on SEC-300 ( 7830 )
Column: ZenixTM SEC-300, ZenixTM - C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM phosphate buffer
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin Cysteine ADC with cleavable linker 2.5 mg/mL
mAU
120
100
80
60
40
ZenixTM - C SEC-300
20
ZenixTM SEC-300
0
2
4
6
8
7
10
12
14
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Herceptin Cysteine ADC Analysis on SEC-300 ( 7830 )
Column: ZenixTM - C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: as indicated
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 10 µL; Samples: Herceptin Cysteine ADC with cleavable linker 2.5 mg/mL
mAU
25
mAU
1. 150 mM phosphate, pH 7.0
2. Buffer 1: 100% IPA = 95%:5%
3. Buffer 1:100% IPA = 90%:10%
120
100
80
60
40
20
20
0
4
6
8
10
12
min
15
10
5
0
5
6
7
8
8
9
10
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Antibody drug conjugate Analysis on SEC-300 ( 7830 )
Phase comparison
TM
TM
Column: Zenix SEC-300, Zenix -C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM phosphate buffer
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 20 µL; Samples: 1.68 mg/mL ADC
mAU
50
40
30
20
10
Zenix-C
Zenix
0
2
4
6
8
10
12
14 min
Zenix-C SEC-300 minimizes the secondary interaction between ADC and resin.
9
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Antibody drug conjugate Analysis on ZenixTM-C SEC-300 ( 7830 )
Column: ZenixTM-C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: As indicated
Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃
Injection volume: 20 µL; Samples: 1.68 mg/mL ADC
mAU
70 A.
60
150 mM phosphate buffer, pH 7.0
A + 0.2 M arginine, pH 7.0
A : Acetonitrile = 90:10 (v/v)
50 mM Phosphate buffer + 0.2 M NaClO4,
pH 7.0
B.
C.
D.
50
Buffer
R
Tailing
% agg
%
monomer
A
1.58
1.31
4.26
95.74
B
1.54
1.33
3.92
96.08
C
1.5
1.27
4.01
95.99
D
1.73
1.14
4.85
95.15
40
30
20
A
B
10
C
0
D
4
6
8
10
12
min
With 10% acetonitrile and 200 mM NaClO4, total protein recovery, resolution and tailing factor of
monomer peak are improved.
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10
Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
MAb F Analysis on Zenix-300 and Zenix-C 300
Column: Zenix-300, Zenix-C 300 ( 3 µm, 300 Å, 7.8 x 300 mm )
Mobile phase: 150 mM Sodium Phosphate, pH 7.0, Flow rate: 1.0 mL/min
Detector: UV 280 nm, Column temperature: 25 ℃, Pressure: 74 bar
Injection volume: 10 µL, Sample: 1.23 mg/mL MAb F in 10 mM sodium succinate, pH 5.0
mAU
22.5
20
Zenix-C 300
MAb MW 145kD
pI = 8.4
17.5
15
12.5
10
7.5
5
Zenix-300
2.5
0
5
6
7
C-line SEC outperforms
8
9
10
11
12
13 min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Fusion Protein Analysis on SEC-300 ( 7830 )
Column: ZenixTM SEC-300, ZenixTM-C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM Phosphate buffer ( pH 7.0 ) + 200 mM NaCl;
Flow rate: 0.5 mL/min; Detector: UV 214 nm; Column temperature: 25 ℃;
Injection volume: 10 µL; Samples: 1 mg/mL fusion protein, MW 170 kD, pI 6.8-7.0
mAU
Blue: ZenixTM SEC-300
Red: ZenixTM-C SEC-300
350
300
250
200
150
100
50
0
0
5
10
15
20
C-line SEC minimizes the secondary interaction.
12
25
min
Fusion Protein Analysis on ZenixTM-C SEC-300 ( 7830 )
Mobile phase effect
Column: ZenixTM-C SEC-300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: as indication
Flow rate: 0.5 mL/min; Detector: UV 214 nm; Column temperature: 25 ℃;
Injection volume: 10 µL; Samples: 1 mg/mL fusion protein, MW 170 kD, pI 6.8-7.0
mAU
1.
2.
3.
350
150 mM Phosphate buffer , pH 7.0 + 200 mM NaCl
150 mM phosphate, pH 7.0 +10% ACN
150 mM phosphate, pH 7.0
300
250
200
150
100
1
2
3
50
0
0
5
10
15
20
25
30
min
10% retention time offset for presentation purpose
13
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
2 Glycoprotein I analysis on Zenix-300 and Zenix-C 300
Column: 7.8x300 mm, Mobile phase: 150 mM Sodium Phosphate Buffer pH 7.0, Flow
rate: 1.0 mL/min, Sample: 2 Glycoprotein I (0.5 mg/mL in 1M glycine, 750 mM NaCl,
pH 7.4), Injection volume: 20 mL, Detection: UV 280 nm
mAU
20
17.5
15
12.5
10
7.5
Zenix SEC-300
5
2.5
Zenix-C SEC-300
0
4
6
8
10
12
min
Zenix-C gives a better separation
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
mPEG-peptide (20 kD PEG + 4 kD peptide)
Mobile phase is 150 mM phosphate buffer, pH 7.0. Flow rate is at 1.0 mL/min with UV214
nm detection. mPEG-peptide concentration is 6 mg/mL, injection volume is 10µL.
mAU
Traditional SEC
120
80
40
0
mAU
Zenix-C SEC
400
mPEG-peptide
300
200
2xmPEG-peptide
100
0
2
4
6
8
10
12
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Pegylated Exenatide on Zenix-300 and Zenix-C 300
Column: Zenix-300 and Zenix-C 300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 50 mM CH3COONH4 : ACN = 90 : 10 ( v/v ),
Flow rate: 0.5 mL/min
Detector: UV 214 nm, Column temperature: 25 ℃, Injection volume: 15 µL
Pressure: 42 bar, Sample: 3.3 mg/mL PEG-Exanatide in water (PEG 23 kD)
mAU
500
Tailing 1.07
450
400
350
300
250
200
Zenix-C 300
150
Tailing 2.37
Zenix 300
100
10
12
14
16
18
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Angiotensin I Acetate separation on Zenix-80 and Zenix-C 80
Column: Zenix SEC-80 7.8x300 mm, Flow rate: 1mL/min, Detection: UV 214 and 280 nm,
Mobile phase: 150 mM Sodium Phosphate Buffer pH 7.0, Injection Volume: 5 µL
mAU
Zenix SEC-80
Zenix-C SEC-80
350
300
250
200
150
100
50
0
6
8
10
12
14
16
18
min
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Membrane protein Aqpz separation
on Zenix-300 and Zenix-C 300
Column: SRT-C 300, 7.8x300, Mobile phase: 20 mM TrisHCl, pH 7.0, 190 mM NaCl, 10 mM
KCl, 40 mM Octyl glucoside, Detection: 280nm, Flow: 1 mL/min, Injection: 2 mL of 6 mg/mL
mAU
Aqpz is a 23kD protein, BOG is nonionic.
50
40
30
Zenix-300
20
10
SRT-C 300
0
4
6
8
10
12
min
SRT-C 300 gives the best separation with less secondary interaction, baseline
separation with the high molecular weight proteins.
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Bacterial K Channel (16 kD homotetramer) in 0.261% DDM
Column dimension: 4.6 x 300 mm, Mobile phase: 20 mM Tris pH 7.5, 20 mM NaCl, 0.261% DDM,
Detection: 280 nm, Flow rate: 0.35 mL/min, AKTA FPLC system
SRT®-C SEC has the best recovery for Bacterial K Channel in the presence of detergents.
mAU
25
SRT -C SEC
20
15
10
Traditional SEC
5
0
2
4
6
8
10
12
min
Acknowledgement: Sung Lee at Scripps.
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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

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