The Challenge: Sepax SEC C-line columns!

Transcription

1 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

2 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 3 mm, 5 mm Zenix 8, 1, 15, 3, Zenix-C 8,1, 15, 3 Pore size (Å) SRT 1, 15, 3, 5, 1 & 2 SRT-C 1, 15, 3, 5, 1 & 2 Surface structure Chemically bonded stand-up monolayer Chemically bonded lay-down monlayer Applications 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 SRT SEC-1 (1 mm) is the latest addition for semi-prep and prep scale separation. SRT SEC-1 and 2 are suitable for very large biomolecule separation. 2

3 Herceptin Analysis on Zenix TM SEC-3 ( 783 ) Column: Zenix TM SEC-3, ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: 15 mm phosphate buffer Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 1 µl; Samples: Herceptin 2.34 mg/ml Rs=

4 Herceptin Lysine ADC Analysis on SEC-3 ( 783 ) 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 Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 1 µl; Samples: Herceptin-lysine conjugate 2.5 mg/ml Zenix TM - C SEC-3 Zenix TM SEC

5 Herceptin-Lysine Drug Conjugate Analysis Zenix-C SEC-3 vs. Tosoh TSKgel G3SW XL Column: Zenix - C SEC-3 (3 µm, 3 Å, 7.8 x 3 mm), Tosoh TSKgel G3SW XL (5 µm, 25 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: Herceptin-lysine drug conjugate 2.5 mg/ml 1 Rs=1.45, total area Rs=1.18, Total area Zenix-C Tosoh TSKgel G3SW XL Disclaimer: TSKgel and Tosoh Bioscience are registered trademarks of Tosoh Corporation; Comparative separations may not be representative of all applications.

6 Herceptin Lysine ADC Analysis on SEC-3 ( 783 ) Column: Zenix TM - C SEC-3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: as indicated Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 1 µl; Samples: Herceptin lysine conjugate 2.5 mg/ml mm phosphate, ph 7. Rs=1.41, tailing= Buffer 1: 1% IPA = 95%:5% Rs=1.5, tailing= Buffer 1:1% IPA = 9%:1% Rs=1.45, tailing=

7 Herceptin Cysteine ADC (cleavable linker) Analysis on SEC-3 ( 783 ) 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 Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 1 µl; Samples: Herceptin Cysteine ADC with cleavable linker 2.5 mg/ml Zenix TM - C SEC-3 Zenix TM SEC

8 Herceptin Cysteine ADC Analysis on SEC-3 ( 783 ) Column: Zenix TM - C SEC-3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: as indicated Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 1 µl; Samples: Herceptin Cysteine ADC with cleavable linker 2.5 mg/ml mm phosphate, ph Buffer 1: 1% IPA = 95%:5% 3. Buffer 1:1% IPA = 9%:1%

9 Antibody drug conjugate Analysis on SEC-3 ( 783 ) 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 Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 2 µl; Samples: 1.68 mg/ml ADC Zenix-C Zenix Zenix-C SEC-3 imizes the secondary interaction between ADC and resin. 9

10 Antibody drug conjugate 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: As indicated Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 Injection volume: 2 µl; Samples: 1.68 mg/ml ADC A. 15 mm phosphate buffer, ph 7. B. A +.2 M arginine, ph 7. C. A : Acetonitrile = 9:1 (v/v) D. 5 mm Phosphate buffer +.2 M NaClO 4, ph 7. Buffer R Tailing % agg % monomer A B C D A B 1 C D With 1% acetonitrile and 2 mm NaClO 4, total protein recovery, resolution and tailing factor of monomer peak are improved. 1

11 MAb F Analysis on Zenix-3 and Zenix-C 3 Column: Zenix-3, Zenix-C 3 ( 3 µm, 3 Å, 7.8 x 3 mm ) Mobile phase: 15 mm Sodium Phosphate, ph 7., Flow rate: 1. ml/ Detector: UV 28 nm, Column temperature: 25, Pressure: 74 bar Injection volume: 1 µl, Sample: 1.23 mg/ml MAb F in 1 mm sodium succinate, ph MAb MW 145kD pi = 8.4 Zenix-3 Zenix-C C-line SEC outperforms

12 Fusion Protein Analysis on SEC-3 ( 783 ) 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. 12

13 Fusion Protein Analysis on Zenix TM -C SEC-3 ( 783 ) 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 mm Phosphate buffer, ph mm NaCl mm phosphate, ph 7. +1% ACN mm phosphate, ph % retention time offset for presentation purpose 13

14 2 Glycoprotein I analysis on Zenix-3 and Zenix-C 3 Column: 7.8x3 mm, Mobile phase: 15 mm Sodium Phosphate Buffer ph 7., Flow rate: 1. ml/, Sample: 2 Glycoprotein I (.5 mg/ml in 1M glycine, 75 mm NaCl, ph 7.4), Injection volume: 2 ml, Detection: UV 28 nm Zenix SEC-3 Zenix-C SEC Zenix-C gives a better separation

15 mpeg-peptide (2 kd PEG + 4 kd peptide) Mobile phase is 15 mm phosphate buffer, ph 7.. Flow rate is at 1. ml/ with UV214 nm detection. mpeg-peptide concentration is 6 mg/ml, injection volume is 1µL. Traditional SEC Zenix-C SEC mpeg - peptide 2 1 2xmPEG - peptide

16 Pegylated Exenatide on Zenix-3 and Zenix-C 3 Column: Zenix-3 and Zenix-C 3 ( 3 µm, 3 Å, 7.8 x 3 mm) Mobile phase: 5 mm CH 3 COONH 4 : ACN = 9 : 1 ( v/v ), Flow rate:.5 ml/ 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) Tailing Zenix-C 3 Zenix 3 Tailing

17 Angiotensin I Acetate separation on Zenix-8 and Zenix-C 8 Column: Zenix SEC-8 7.8x3 mm, Flow rate: 1mL/, Detection: UV 214 and 28 nm, Mobile phase: 15 mm Sodium Phosphate Buffer ph 7., Injection Volume: 5 µl 35 Zenix SEC-8 Zenix-C SEC

18 Membrane protein Aqpz separation on Zenix-3 and Zenix-C 3 Column: SRT-C 3, 7.8x3, Mobile phase: 2 mm TrisHCl, ph 7., 19 mm NaCl, 1 mm KCl, 4 mm Octyl glucoside, Detection: 28nm, Flow: 1 ml/, Injection: 2 ml of 6 mg/ml Aqpz is a 23kD protein, BOG is nonionic Zenix-3 1 SRT-C SRT-C 3 gives the best separation with less secondary interaction, baseline separation with the high molecular weight proteins.

19 Bacterial K Channel (16 kd homotetramer) in.261% DDM Column dimension: 4.6 x 3 mm, Mobile phase: 2 mm Tris ph 7.5, 2 mm NaCl,.261% DDM, Detection: 28 nm, Flow rate:.35 ml/, AKTA FPLC system SRT -C SEC has the best recovery for Bacterial K Channel in the presence of detergents SRT -C SEC Traditional SEC Acknowledgement: Sung Lee at Scripps.