BSA pegylation PG Journal of chromatographic A, 1147 (2007)

Transcription

1 BSA pegylation BSA 5mg/mL, pi= 4.7, Sigma A796, 65-7% free sulfyhydryl, cysteine 34 of BSA is not paired with another cysteine in the protein structure 1. PEG 1kD, kd, 3 kd 1mg/mL PEG:BSA=1.2:1 (molar ratio) Incubate at RT for overnight Columns:, Zenix SEC-3 and SRT-C SEC-5, Proteomix SAX 1. Journal of chromatographic A, 1147 (7)

2 PEG PEG-1kD PEG-kD PEG-3kD From NOF America, SUNBRIGHT MA Series (Maleimide PEG), target group -SH 2

3 4-12% Bis-Tris SDS-PAGE-MOPS buffer KD Marker 2. BSA control 3. BSA + 1 kd PEG 4. BSA + kd PEG 5. BSA + 3 kd PEG Due to the non-reducing condition, the MW of BSA samples appear lower which allows the identification of covalently linked aggregates. 3

4 Pegylated BSA (1 kd PEG) Analysis on SEC-3 ( 783 ) Column: Zenix TM 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 ; Samples: BSA control (5 mg/ml in sodium acetate), 1 ml PEG 1 kd, 1 mg/ml, 5 ml 1 kd pegylated BSA, 4.59 mg/ml, 11 ml 1 8 Green: 1 kd Pegylated BSA Red: BSA control Blue: 1 kd PEG control BSA monomer 6 Pegylated BSA monomer Pegylated BSA aggregate

5 Pegylated BSA ( kd PEG) Analysis on SEC-3 ( 783 ) Column: Zenix TM 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 ; Samples: BSA control (5 mg/ml in sodium acetate), 1 ml PEG kd, 1 mg/ml, 5 ml kd pegylated BSA, 4.23 mg/ml, 12 ml 1 8 Green: kd Pegylated BSA Red: BSA control Blue: kd PEG control BSA monomer 6 Pegylated BSA monomer Pegylated BSA aggregate BSA dimer

6 Pegylated BSA (3 kd PEG) Analysis on SEC-3 ( 783 ) Column: Zenix TM 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 ; Samples: BSA control (5 mg/ml in sodium acetate), 1 ml PEG 3 kd, 1 mg/ml, 5 ml 3 kd pegylated BSA, 3.93 mg/ml, 12.7 ml 1 8 Green: 3 kd Pegylated BSA Red: BSA control Blue: 3 kd PEG control BSA monomer 6 Pegylated BSA aggregate Pegylated BSA monomer BSA dimer

7 Duplicated injection- Pegylated BSA (3 kd PEG) Column: Zenix TM 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 ; Samples: BSA control (5 mg/ml in sodium acetate), 1 ml PEG 3 kd, 1 mg/ml, 5 ml 3 kd pegylated BSA, 3.93 mg/ml, 12.7 ml Injection 1 Injection

8 Pegylated BSA overlays with QC standard Column: Zenix TM 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 ; Samples: Protein standards, Thyroglobulin (67 kd), BSA (66 kd), Ribonuclease A (14 kd), Uracil (1 Da) 1,, 3 kd pegylated BSA, 5 mg each kd PEG kd PEG 1 kd PEG STD 67 kd 132 kd 66 kd 14 kd Da 8 8

9 Pegylated BSA analysis with two Zenix in tandem Column: 2 x Zenix TM SEC-3, ( 3 µm, 3 Å, 7.8 x 3 mm), SN Mobile phase: 15 mm Phosphate buffer, ph 7.; Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 ; Samples: QC standards, 1,, 3 kd pegylated BSA, 5 mg each 5 1 Da 3 3 kd PEG 14 kd 1 kd PEG 1 kd PEG STD 67 kd 132 kd 66 kd

10 Pegylated (3 kd PEG) BSA analysis with two Zenix in tandem Column: 2 x Zenix TM SEC-3, ( 3 µm, 3 Å, 7.8 x 3 mm), SN Mobile phase: 15 mm Phosphate buffer, ph 7.; Flow rate: 1 ml/; Detector: UV 28 nm; Column temperature: 25 ; Samples: BSA 5 mg, 3 kd pegylated BSA, 5 mg each kd pegylated BSA Unpegylated BSA

11 Pegylated (3 kd PEG) BSA analysis Pore size difference Column: Zenix TM SEC-3, (3 µm, 3 Å, 7.8 x 3 mm), SRT SEC-5 (5 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 ; Samples: 3 kd pegylated BSA, 5 mg Zenix TM SEC-3 SRT SEC

12 Pegylated (3 kd PEG) BSA analysis Pore size difference Column: Zenix TM SEC-3, (3 µm, 3 Å, 7.8 x 3 mm), SRT SEC-5 (5 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 ; Samples: 1,, 3 kd Maleimide PEG (sunbright from NOF), 5 mg 16 1 kd kd kd Zenix SEC-3 SRT SEC

13 Proteomix SAX Pegylated BSA separation-4615 Column: Proteomix SAX-NP5 ( 5 µm, 4.6 x 15 mm), Mobile phase: A: Bis-Tris Propane, ph 7., B: A + 1 M NaCl, ph 7., Flow rate:.8 ml/, 17 bar; Detector: UV 28 nm, Column temperature: 25, Samples: 5 mg each, Unconjugated BSA, PEG 1 kd, PEG kd and PEG 3 kd conjugated BSA 5 Time %B 3 2% 21 3% 3 1 BSA-PEG 3 kd BSA-PEG kd BSA-PEG 1 kd BSA

14 Proteomix SAX Pegylated BSA separation Column: Proteomix SAX-NP5 ( 5 µm, 4.6 x 25 mm), Mobile phase: A: Bis-Tris Propane, ph 7., B: A + 1 M NaCl, ph 7., Flow rate:.8 ml/, 17 bar; Detector: UV 28 nm, Column temperature: 25, Samples: 15 mg each, Unconjugated BSA, PEG 1 kd, PEG kd and PEG 3 kd conjugated BSA Time %B 5 2% 35 3% BSA-PEG 3 kd BSA-PEG kd BSA-PEG 1 kd BSA

15 Proteomix SAX 1 kd Pegylated BSA Loading Column: Proteomix SAX-NP5 ( 5 µm, 4.6 x 25 mm), Mobile phase: A: Bis-Tris Propane, ph 7., B: A + 1 M NaCl, ph 7., Flow rate:.8 ml/, 17 bar; Detector: UV 28 nm, Column temperature: 25, Samples: 5, 15 mg each, PEG 1 kd conjugated BSA mg 5 mg

16 Proteomix SAX kd Pegylated BSA Loading Column: Proteomix SAX-NP5 ( 5 µm, 4.6 x 25 mm), Mobile phase: A: Bis-Tris Propane, ph 7., B: A + 1 M NaCl, ph 7., Flow rate:.8 ml/, 17 bar; Detector: UV 28 nm, Column temperature: 25, Samples: 5, 15 mg each, PEG kd conjugated BSA mg 5 mg

17 Proteomix SAX 3 kd Pegylated BSA Loading Column: Proteomix SAX-NP5 ( 5 µm, 4.6 x 25 mm), Mobile phase: A: Bis-Tris Propane, ph 7., B: A + 1 M NaCl, ph 7., Flow rate:.8 ml/, 17 bar; Detector: UV 28 nm, Column temperature: 25, Samples: 5, 1, 15 mg each, PEG 3 kd conjugated BSA mg 1 mg 5 mg

18 SAX collected fractions on Zenix SEC-3 peak 1 and 2 from previous slide Column: Zenix TM 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 ; Samples: 3 kd pegylated BSA fractionated on SAX, fraction 1 and 2 concentrated with a 3kD Amicon Ultra kd PEG-BSA mixture Fraction 2 Fraction