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) 172-182
PEG PEG-1kD PEG-kD PEG-3kD From NOF America, SUNBRIGHT MA Series (Maleimide PEG), target group -SH 2
4-12% Bis-Tris SDS-PAGE-MOPS buffer KD 116 97 66 55 36 31 21 1. Marker 2. BSA control 3. BSA + 1 kd PEG 4. BSA + kd PEG 5. BSA + 3 kd PEG 14 1 2 3 4 5 Due to the non-reducing condition, the MW of BSA samples appear lower which allows the identification of covalently linked aggregates. 3
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 6 7 8 9 1 11 4 4
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 5 6 7 8 9 1 11 5 5
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 5 6 7 8 9 1 11 6 6
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 7 6 5 3 1 Injection 1 Injection 2 4 5 6 7 8 9 1 11 12 7 7
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 8 7 6 5 3 1 3 kd PEG kd PEG 1 kd PEG STD 67 kd 132 kd 66 kd 14 kd 5 6 7 8 9 1 11 12 1 Da 8 8
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 4499 + 32695 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 7.5 1 12.5 15 17.5 22.5 25 9
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 4499 + 32695 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 6 5 3 1 3 kd pegylated BSA Unpegylated BSA 6 8 1 12 14 16 18 22 24 1 1
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 6 5 3 1 Zenix TM SEC-3 SRT SEC-5 4 5 6 7 8 9 1 11 12 13 11 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: 1,, 3 kd Maleimide PEG (sunbright from NOF), 5 mg 16 1 kd 1 1 1 8 6 3 kd kd Zenix SEC-3 SRT SEC-5 4 5 6 7 8 9 1 11 12 13 12 12
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 5 1 15 25 13 13
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 8 7 6 5 3 1 Time %B 5 2% 35 3% BSA-PEG 3 kd BSA-PEG kd BSA-PEG 1 kd BSA 5 1 15 25 3 35 14 14
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 35 3 25 15 1 5 15 mg 5 mg 5 1 15 25 3 35 15 15
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 35 3 25 15 1 5 15 mg 5 mg 5 1 15 25 3 35 16 16
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 35 3 25 2 15 1 5 1 15 mg 1 mg 5 mg 5 1 15 25 3 35 17 17
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 7 6 5 3 1 3 kd PEG-BSA mixture Fraction 2 Fraction 1 2 4 6 8 1 12 18