NOTES. Fusion Protein Separation. Highlighted FACTS:

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1 Fusion Protein Separation NOTES Highlighted FACTS: Fusion proteins are structural hybrids that contain two different proteins, which retain the functional properties from the original proteins. Zenix and Zenix- C SEC are suitable for aggregation/monomer separation of fusion proteins including Fc fusion and HSA fusion proteins. Fc Fusion Protein 1 - Mobile Phase Effect Column: Zenix TM SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm); Mobile phase 1: 15 mm Phosphate buffer, ph 7.; Mobile phase 2: 5mM phosphate buffer, 15 mm NaCl, 27 mm L- Arginine, ph 7.; Flow rate:.5 ml/; Detector: UV 28 nm; Column temperature: Room temperature; Injection volume: 5 µl; Pressure: 5 bar; Sample: CHO cell expressed Fc- fusion protein 16 kd ( 2. mg/ml ). 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. Fc Fusion Protein 2: rhtnfr- Fc - SEC Phase Comparison Column: Zenix TM SEC- 3 (3 µm, 3 Å, 7.8 x 3 mm); Zenix TM - C SEC- 3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Phosphate buffer, ph 7.; Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25, Injection volume: 1 µl; Samples: Recombinant human tumor necrosis factor receptor- Fc ( rhtnfr- Fc ) 2 mg/ml in water. Fusion Protein 3 - Mobile Phase Effect Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: as indication; Flow rate:.5 ml/; Detector: UV 214 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: 1 mg/ml fusion protein, MW 17 kd, pi % retention time offset for presentation purpose. Fusion Protein 3 - SEC Phase comparison Column: Zenix TM SEC- 3, Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm Phosphate buffer ( ph 7. ) + 2 mm NaCl; Flow rate:.5 ml/; Detector: UV 214 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: 1 mg/ml fusion protein, MW 17 kd, pi tech.com info@sepax- tech.com Toll Free: SEPAX- US 1

2 Fusion Protein Separation Fusion Protein 4 - Mobile Phase Effect Column: Zenix TM SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm); Mobile phase 1: 25 mm Na2HPO4, 2 mm NaCl, ph 6.8; Mobile phase 2: 15 mm Phosphate buffer, ph 7.; Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25 ; Pressure: 82 bar; Injection volume: 2 µl; Sample: 1 mg/ml Fusion protein (15 kd, pi 5., 5 mg/ml, dilute to 1 mg/ml). NOTES Fusion Protein 4 - Column Length Effect Column: Zenix TM SEC- 3 (3 µm, 3 Å), 7.8 x 3 mm and 7.8 x 5 mm+ 7.8 x 3 mm; Mobile phase: 25 mm Na2HPO4, 2 mm NaCl, ph 6.8; Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25 ; Pressure: 9 bar; Injection volume: 2 µl; Sample: 1 mg/ml Fusion protein (15 kd, pi 5., 5 mg/ml, dilute to 1 mg/ml). HSA Conjugated Peptide - SEC Phase Comparison Column: Zenix TM - C SEC- 3, Zenix TM SEC- 3 (3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: A. 15 mm Phosphate buffer, ph 7.; Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment). HSA Fusion Protein - Mobile Phase Effect Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: A. 15 mm Phosphate buffer, ph 7., B: 15 mm; Phosphate buffer ( ph 7. ) : IPA = 95 : 5 ( v/v ); Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment). HSA Fusion Protein vs HSA Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm); Mobile phase: 15 mm; Phosphate buffer ( ph 7. ) : IPA = 95 : 5 ( v/v ); Flow rate: 1. ml/; Detector: UV 28 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., 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 25 mm); Mobile phase: Piperazine, Triethanolae, bis- tris propane and N- methylpiperazine, 2 mm each,a, ph 9.7; B, ph 3. ( adjust by HCl ); Flow rate:.5 ml/; Detector: UV 28 nm; Column temperature: 3 ; Pressure: 139 bar; Injection volume: 1 µl, MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment. tech.com info@sepax-tech.com Toll Free: SEPAX- US 2

3 ZP19 Analysis of Fc fusion protein on Zenix vs. SRT Zenix SEC-3 SRT SEC Columns: SRT SEC-3 (5 µm, 3 Ǻ, 7.8 x 3 mm), Zenix SEC-3 (3 µm, 3 Ǻ, 7.8 x 3 mm) Mobile Phase: 15 mm sodium phosphate buffer, ph 7. Flow Rate: 1. ml/; Temperature: Ambient Detection: UV 214 nm Injection Volume: 2 µl Samples: Recombinant tumor necrosis factor receptor-fc fusion protein (rhtnfr-fc), including oligomers (44 kd and 36 kd), monomer (15 kd), and impurity (12 kd). Keywords: Size exclusion, SRT, Zenix, protein, Recombinant tumor necrosis factor receptor-fc fusion protein, high resolution Sepax Technologies, Inc.

4 Fusion Protein Analysis on SEC- 3 ( 783 ) EP11 Column: Zenix TM SEC- 3, Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: 15 mm Phosphate buffer ( ph 7. ) + 2 mm NaCl; Flow rate:.5 ml/; Detector: UV 214 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: 1 mg/ml fusion protein, MW 17 kd, pi Blue: Zenix TM SEC- 3 Red: Zenix TM - C SEC C-line SEC imizes the secondary interaction.

5 Fusion Protein Analysis on Zenix TM - C SEC- 3 ( 783 ) Mobile phase effect EP11 Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: as indication Flow rate:.5 ml/; Detector: UV 214 nm; Column temperature: 25 ; Injection volume: 1 µl; Samples: 1 mg/ml fusion protein, MW 17 kd, pi mm Phosphate buffer, ph mm NaCl mm phosphate, ph 7. +1% ACN mm phosphate, ph % retention time offset for presentation purpose

6 Fusion Protein Analysis on Zenix TM - C SEC- 3 ( 783 ) Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: 15 mm Phosphate buffer ( ph 7. ) + 2 mm NaCl, Flow rate: 1. ml/, Detector: UV 214 nm, Column temperature: 25, Injection volume: 1 µl, Samples: 1 mg/ml fusion protein Pressure: 76 bar EP

7 HSA fusion protein separation on Zenix- C SEC- 3 EP13 Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: A. 15 mm Phosphate buffer, ph 7., B: 15 mm Phosphate buffer ( ph 7. ) : IPA = 95 : 5 ( v/v ), Flow rate: 1. ml/, Detector: UV 28 nm, Column temperature: 25, Injection volume: 1 µl, Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment) B A R=1.58 R=

8 HSA fusion protein separation on Zenix- C SEC- 3 Column: Zenix TM - C SEC- 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: 15 mm Phosphate buffer ( ph 7. ) : IPA = 95 : 5 ( v/v ), Flow rate: 1. ml/, Detector: UV 28 nm, Column temperature: 25, Injection volume: 1 µl, Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment), HSA 2 mg/ml EP HSA fusion protein HSA

9 HSA fusion protein separation on Zenix and Zenix- C Column: Zenix TM - C SEC- 3, Zenix TM SEC- 3 (3 µm, 3 Å, 7.8 x 3 mm), Mobile phase: A. 15 mm Phosphate buffer, ph 7., Flow rate: 1. ml/, Detector: UV 28 nm, Column temperature: 25, Injection volume: 1 µl, Samples: HSA fusion peptide 5 mg/ml (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment) EP Zenix TM - C SEC- 3 Zenix TM SEC

10 HSA fusion protein Analysis on Proteomix SAX- NP5 PP121 Column: Proteomix SAX- NP5 (5 µm, 4.6 x 25 mm) Mobile phase: Piperazine, Triethanolae, bis- tris propane and N- methylpiperazine, 2 mm each, A, ph 9.7; B, ph 3. ( adjust by HCl ) Flow rate:.5 ml/, Detector: UV 28 nm, Column temperature: 3, Pressure: 139 bar Sample: HSA fusion peptide 5 mg/ml, 1 µl (MW 75 kd, pi 5., HSA conjugated peptide in diabetes treatment) Time Flow A% B% ( ) ( ml/) HSA, 2 mg/ml, 1 µl E2HSA, 5 mg/ml, 1 µl HSA+E2HSA (1:1 v/v), 3 µl

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