Expression of polyhedrin-hEGF fusion protein in cultured ...
Fusion Protein Separation NOTES - obrnutafaza.hr · rtech.com info@sepax tech.com Toll Free: 1...
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www.sepaxtech.com [email protected] Toll Free: 1877SEPAXUS 1 NOTES Fusion Protein Separation Fc Fusion Protein 1 Mobile Phase Effect Column: Zenix TM SEC300 ( 3 µm, 300 Å, 7.8 x 300 mm); Mobile phase 1: 150 mM Phosphate buffer, pH 7.0; Mobile phase 2: 50mM phosphate buffer, 150 mM NaCl, 27 mM LArginine, pH 7.0; Flow rate: 0.5 mL/min; Detector: UV 280 nm; Column temperature: Room temperature; Injection volume: 50 µL; Pressure: 50 bar; Sample: CHO cell expressed Fcfusion protein 160 kD ( 2.0 mg/mL ). Fc Fusion Protein 2: rhTNFRFc SEC Phase Comparison Column: Zenix TM SEC300 (3 µm, 300 Å, 7.8 x 300 mm); Zenix TM C SEC300 (3 µm, 300 Å, 7.8 x 300 mm); Mobile phase: 150 mM Phosphate buffer, pH 7.0; Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃, Injection volume: 10 µL; Samples: Recombinant human tumor necrosis factor receptorFc ( rhTNFRFc ) 2 mg/mL in water. Fusion Protein 3 Mobile Phase Effect Column: Zenix TM C SEC300 ( 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 6.87.0 10% retention time offset for presentation purpose. Fusion Protein 3 SEC Phase comparison Column: Zenix TM SEC300, Zenix TM C SEC300 ( 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 6.87.0. ¾ Fusion proteins are structural hybrids that contain two different proteins, which retain the functional properties from the original proteins. ¾ Zenix and ZenixC SEC are suitable for aggregation/monomer separation of fusion proteins including Fc fusion and HSA fusion proteins. Highlighted FACTS: ¾ Proteomix SAX and SCX provide high resolution separation of fusion proteins, especially different glycosylation forms, charge variants and fusion proteins from their formulation components. *Please click above highlights for more product information.
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Transcript of Fusion Protein Separation NOTES - obrnutafaza.hr · rtech.com info@sepax tech.com Toll Free: 1...
www.sepax-‐tech.com info@sepax-‐tech.com Toll Free: 1-‐877-‐SEPAX-‐US 1
NOTES Fusion Protein Separation
Fc Fusion Protein 1 -‐ Mobile Phase Effect Column: ZenixTM SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm); Mobile phase 1: 150 mM Phosphate buffer, pH 7.0; Mobile phase 2: 50mM phosphate buffer, 150 mM NaCl, 27 mM L-‐Arginine, pH 7.0; Flow rate: 0.5 mL/min; Detector: UV 280 nm; Column temperature: Room temperature; Injection volume: 50 µL; Pressure: 50 bar; Sample: CHO cell expressed Fc-‐fusion protein 160 kD ( 2.0 mg/mL ).
Fc Fusion Protein 2: rhTNFR-‐Fc -‐ SEC Phase Comparison Column: ZenixTM SEC-‐300 (3 µm, 300 Å, 7.8 x 300 mm); ZenixTM-‐C SEC-‐300 (3 µm, 300 Å, 7.8 x 300 mm); Mobile phase: 150 mM Phosphate buffer, pH 7.0; Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃, Injection volume: 10 µL; Samples: Recombinant human tumor necrosis factor receptor-‐Fc ( rhTNFR-‐Fc ) 2 mg/mL in water.
Fusion Protein 3 -‐ 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 10% retention time offset for presentation purpose.
Fusion Protein 3 -‐ SEC Phase comparison 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.
¾ Fusion proteins are structural hybrids that contain two different proteins, which retain the functional properties from the original proteins.
¾ Zenix and Zenix-‐C SEC are suitable for aggregation/monomer separation of fusion proteins including Fc fusion and HSA fusion proteins.
Highlighted FACTS:
¾ Proteomix SAX and SCX provide high resolution separation of fusion proteins, especially different glycosylation forms, charge variants and fusion proteins from their formulation components.
*Please click above highlights for more product information.
www.sepax-‐tech.com info@sepax-tech.com Toll Free: 1-‐877-‐SEPAX-‐US 2
NOTES Fusion Protein Separation
Fusion Protein 4 -‐ Mobile Phase Effect Column: ZenixTM SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm); Mobile phase 1: 25 mM Na2HPO4, 200 mM NaCl, pH 6.8; Mobile phase 2: 150 mM Phosphate buffer, pH 7.0; Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃; Pressure: 82 bar; Injection volume: 2 µL; Sample: 10 mg/mL Fusion protein (150 kD, pI 5.0, 50 mg/mL, dilute to 10 mg/mL).
Fusion Protein 4 -‐ Column Length Effect Column: ZenixTM SEC-‐300 (3 µm, 300 Å), 7.8 x 300 mm and 7.8 x 50 mm+ 7.8 x 300 mm; Mobile phase: 25 mM Na2HPO4, 200 mM NaCl, pH 6.8; Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃; Pressure: 90 bar; Injection volume: 2 µL; Sample: 10 mg/mL Fusion protein (150 kD, pI 5.0, 50 mg/mL, dilute to 10 mg/mL).
HSA Conjugated Peptide -‐ SEC Phase Comparison Column: ZenixTM-‐C SEC-‐300, ZenixTM SEC-‐300 (3 µm, 300 Å, 7.8 x 300 mm); Mobile phase: A. 150 mM Phosphate buffer, pH 7.0; Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃; Injection volume: 10 µL; Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment).
HSA Fusion Protein -‐ Mobile Phase Effect Column: ZenixTM-‐C SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm); Mobile phase: A. 150 mM Phosphate buffer, pH 7.0, B: 150 mM; Phosphate buffer ( pH 7.0 ) : IPA = 95 : 5 ( v/v ); Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃; Injection volume: 10 µL; Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment).
HSA Fusion Protein vs HSA Column: ZenixTM-‐C SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm); Mobile phase: 150 mM; Phosphate buffer ( pH 7.0 ) : IPA = 95 : 5 ( v/v ); Flow rate: 1.0 mL/min; Detector: UV 280 nm; Column temperature: 25℃; Injection volume: 10 µL; Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment), HSA 2 mg/mL.
HSA conjugated peptide Analysis on Proteomix® SAX-‐NP5 Column: Proteomix® SAX-‐NP5 (5 µm, 4.6 x 250 mm); Mobile phase: Piperazine, Triethanolamine, bis-‐tris propane and N-‐methylpiperazine, 20 mM each,A, pH 9.7; B, pH 3.0 ( adjust by HCl ); Flow rate: 0.5 mL/min; Detector: UV 280 nm; Column temperature: 30 ℃; Pressure: 139 bar; Injection volume: 10 µL, MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment.
ZP1009
© Sepax Technologies, Inc.
Columns: SRT® SEC-300 (5 µm, 300 Ǻ, 7.8 x 300 mm), Zenix™ SEC-300 (3 µm, 300 Ǻ, 7.8 x 300 mm)
Mobile Phase: 150 mM sodium phosphate buffer, pH 7.0 Flow Rate: 1.0 mL/min; Temperature: Ambient Detection: UV 214 nm Injection Volume: 20 µL Samples: Recombinant tumor necrosis factor receptor-Fc fusion protein
(rhTNFR-Fc), including oligomers (440 kD and 360 kD), monomer (150 kD), and impurity (120 kD).
Analysis of Fc fusion protein on Zenix™ vs. SRT®
Keywords: Size exclusion, SRT, Zenix, protein, Recombinant tumor necrosis factor receptor-Fc fusion protein, high resolution
min0 2 4 6 8 10 12 14
SRT SEC-300
Zenix SEC-300
1
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
Fusion Protein Analysis on SEC-‐300 ( 7830 )
min0 5 10 15 20 25
mAU
0
50
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350 Blue: ZenixTM SEC-‐300
Red: ZenixTM-‐C SEC-‐300
C-line SEC minimizes the secondary interaction.www.sepax-tech.com
Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.
EP1001
2
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
Fusion Protein Analysis on ZenixTM-‐C SEC-‐300 ( 7830 )Mobile phase effect
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
10% retention time offset for presentation purpose
min0 5 10 15 20 25 30
mAU
0
50
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250
300
3501. 150 mM Phosphate buffer , pH 7.0 + 200 mM NaCl
2. 150 mM phosphate, pH 7.0 +10% ACN
3. 150 mM phosphate, pH 7.0
1
2
3
EP1001
3
Column: 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: 1.0 mL/min, Detector: UV 214 nm, Column temperature: 25 ℃,
Injection volume: 10 µL, Samples: 1 mg/mL fusion protein
Pressure: 76 bar
Fusion Protein Analysis on ZenixTM-‐C SEC-‐300 ( 7830 )
min0 2 4 6 8 10 12 14
mAU
0
50
100
150
200
6.283
7.087
7.955
min4 5 6 7 8 9
mAU
0
20
40
60
80
6.283
7.087
7.955
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
EP1001
HSA fusion protein separation on Zenix-‐C SEC-‐300
min2 4 6 8 10 12
mAU
0
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40
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60
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80
Column: ZenixTM-‐C SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: A. 150 mM Phosphate buffer, pH 7.0, B: 150 mM Phosphate buffer ( pH 7.0 ) : IPA = 95 : 5 ( v/v ),
Flow rate: 1.0 mL/min, Detector: UV 280 nm, Column temperature: 25℃, Injection volume: 10 µL,
Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment)
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
R=1.58
R= 1.09A
B
EP1003
HSA fusion protein separation on Zenix-‐C SEC-‐300
Column: ZenixTM-‐C SEC-‐300 ( 3 µm, 300 Å, 7.8 x 300 mm)
Mobile phase: 150 mM Phosphate buffer ( pH 7.0 ) : IPA = 95 : 5 ( v/v ),
Flow rate: 1.0 mL/min, Detector: UV 280 nm, Column temperature: 25℃, Injection volume: 10 µL,
Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment), HSA
2 mg/mL
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
min4 6 8 10 12
mAU
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HSA fusion protein
HSA
EP1003
HSA fusion protein separation on Zenix and Zenix-‐C
Column: ZenixTM-‐C SEC-‐300, ZenixTM SEC-‐300 (3 µm, 300 Å, 7.8 x 300 mm),
Mobile phase: A. 150 mM Phosphate buffer, pH 7.0,
Flow rate: 1.0 mL/min, Detector: UV 280 nm, Column temperature: 25℃, Injection volume: 10 µL,
Samples: HSA fusion peptide 5 mg/mL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment)
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
min2 4 6 8 10 12 14
mAU
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20
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ZenixTM-‐C SEC-‐300
ZenixTM SEC-‐300
EP1003
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
HSA fusion protein Analysis on Proteomix® SAX-‐NP5
www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.
Column: Proteomix® SAX-‐NP5 (5 µm, 4.6 x 250 mm)
Mobile phase: Piperazine, Triethanolamine, bis-‐tris propane and N-‐methylpiperazine, 20 mM each,
A, pH 9.7; B, pH 3.0 ( adjust by HCl )
Flow rate: 0.5 mL/min, Detector: UV 280 nm, Column temperature: 30 ℃, Pressure: 139 bar
Sample: HSA fusion peptide 5 mg/mL, 10 µL (MW 75 kD, pI 5.0, HSA conjugated peptide in diabetes treatment)
min0 5 10 15 20 25
mAU
0
25
50
75
100
125
150
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200
HSA, 2 mg/mL, 10 µL
E2HSA, 5 mg/mL, 10 µL
HSA+E2HSA (1:1 v/v), 30 µL
Time( min )
Flow( mL/min)
A% B%
00.5
35 6530 0 100
PP1021
