BSA pegylation - Sepax Technologies, Inc. · 2020. 10. 20. · BSA pegylation •BSA 5mg/mL, pI= 4.7, Sigma A7096, 65-70% free sulfyhydryl, cysteine 34 of BSA is not paired with another

BSA pegylation

• BSA 5mg/mL, pI= 4.7, Sigma A7096, 65-70% free sulfyhydryl, cysteine 34 of BSA is not paired with another cysteine in the protein structure1.

• PEG 10kD, 20kD, 30 kD 10mg/mL• PEG:BSA=1.2:1 (molar ratio)• Incubate at RT for overnight• Columns:, Zenix SEC-300 and SRT-C SEC-500,

Proteomix SAX

www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.

1. Journal of chromatographic A, 1147 (2007) 172-182

PG1001

PEG

• PEG-10kD• PEG-20kD• PEG-30kD• From NOF America, SUNBRIGHT® MA Series

(Maleimide PEG), target group -SH


2

PG1001

4-12% Bis-Tris SDS-PAGE-MOPS buffer

1 2 3 4 5


KD200

116

9766

55

36

31

21

14

1. Marker2. BSA control3. BSA + 10 kD PEG4. BSA + 20 kD PEG5. BSA + 30 kD PEG

Due to the non-reducing condition, the MW of BSA samples appear lower which allows the identification of covalently linked aggregates.

3

PG1001

4

Column: ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm) Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: BSA control (5 mg/mL in sodium acetate), 10 mL

PEG 10 kD, 10 mg/mL, 5 mL10 kD pegylated BSA, 4.59 mg/mL, 11 mL

Pegylated BSA (10 kD PEG) Analysis on SEC-300 ( 7830 )


min5 6 7 8 9 10 11

mAU

0

20

40

60

80

100Green: 10 kD Pegylated BSARed: BSA controlBlue: 10 kD PEG control

BSA monomer

Pegylated BSA monomer

Pegylated BSA aggregate

4

PG1001

5


PEG 20 kD, 10 mg/mL, 5 mL20 kD pegylated BSA, 4.23 mg/mL, 12 mL



min5 6 7 8 9 10 11

mAU

0

20

40

60

80


BSA monomer

BSA dimer



5

PG1001

6


PEG 30 kD, 10 mg/mL, 5 mL30 kD pegylated BSA, 3.93 mg/mL, 12.7 mL



min5 6 7 8 9 10 11

mAU

0

20

40

60

80


BSA monomer

BSA dimer



6

PG1001

7


PEG 30 kD, 10 mg/mL, 5 mL30 kD pegylated BSA, 3.93 mg/mL, 12.7 mL

Duplicated injection- Pegylated BSA (30 kD PEG)


min4 5 6 7 8 9 10 11 12

mAU

0

10

20

30

40

50

60

70

Injection 1

Injection 2

7

PG1001

8

Column: ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm) Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: Protein standards, Thyroglobulin (670 kD), BSA (66 kD), Ribonuclease A (14 kD), Uracil (120 Da)

10, 20 , 30 kD pegylated BSA, 50 mg each

Pegylated BSA overlays with QC standard


min5 6 7 8 9 10 11 12

mAU

0

10

20

30

40

50

60

70

80

670 kD 132 kD 66 kD 14 kD

120 Da

30 kD PEG20 kD PEG10 kD PEG

STD

8

PG1001

Pegylated BSA analysis with two Zenix in tandemColumn: 2 x ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm), SN 44909 + 32695Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: QC standards, 10, 20, 30 kD pegylated BSA, 50 mg each


min7.5 10 12.5 15 17.5 20 22.5 25

mAU

0

10

20

30

40

50

30 kD PEG

20 kD PEG

10 kD PEG

STD 670 kD 132 kD 66 kD

14 kD

120 Da

9

PG1001

10

Column: 2 x ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm), SN 44909 + 32695Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: BSA 50 mg, 30 kD pegylated BSA, 50 mg each

Pegylated (30 kD PEG) BSA analysis with two Zenix in tandem


min6 8 10 12 14 16 18 20 22 24

mAU

0

10

20

30

40

50

60

Unpegylated BSA

30 kD pegylated BSA

10

PG1001

11

Column: ZenixTM SEC-300, (3 µm, 300 Å, 7.8 x 300 mm), SRT SEC-500 (5 µm, 300 Å, 7.8 x 300 mm)Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: 30 kD pegylated BSA, 50 mg

Pegylated (30 kD PEG) BSA analysis – Pore size difference


min4 5 6 7 8 9 10 11 12 13

mAU

0

10

20

30

40

50

60

ZenixTM SEC-300SRT® SEC-500

11

PG1001

12

Column: ZenixTM SEC-300, (3 µm, 300 Å, 7.8 x 300 mm), SRT SEC-500 (5 µm, 300 Å, 7.8 x 300 mm)Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: 10, 20, 30 kD Maleimide PEG (sunbright from NOF), 50 mg

Pegylated (30 kD PEG) BSA analysis – Pore size difference


min4 5 6 7 8 9 10 11 12 13

mAU

0

20

40

60

80

100

120

140

160

30 kD

20 kD

10 kD

Zenix SEC-300

SRT SEC-500

12

PG1001

13www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Proteomix® SAX – Pegylated BSA separation-4615Column: Proteomix® SAX-NP5 ( 5 µm, 4.6 x 150 mm), Mobile phase: A: Bis-Tris Propane, pH 7.0, B: A + 1 M NaCl, pH 7.0,Flow rate: 0.8 mL/min, 170 bar; Detector: UV 280 nm, Column temperature: 25 ℃,Samples: 50 mg each, Unconjugated BSA, PEG 10 kD, PEG 20 kD and PEG 30 kD conjugated BSA

13

min5 10 15 20 25

mAU

0

10

20

30

40

50

BSA

BSA-PEG 10 kD

BSA-PEG 20 kD

BSA-PEG 30 kD

Time %B

3 2%

21 30%

PG1001


Proteomix® SAX – Pegylated BSA separationColumn: Proteomix® SAX-NP5 ( 5 µm, 4.6 x 250 mm), Mobile phase: A: Bis-Tris Propane, pH 7.0, B: A + 1 M NaCl, pH 7.0,Flow rate: 0.8 mL/min, 170 bar; Detector: UV 280 nm, Column temperature: 25 ℃,Samples: 150 mg each, Unconjugated BSA, PEG 10 kD, PEG 20 kD and PEG 30 kD conjugated BSA

min0 5 10 15 20 25 30 35

mAU

0

10

20

30

40

50

60

70

80

BSA

BSA-PEG 10 kD

BSA-PEG 20 kD

BSA-PEG 30 kD

14

Time %B

5 2%

35 30%

PG1001


Proteomix® SAX – 10 kD Pegylated BSA LoadingColumn: Proteomix® SAX-NP5 ( 5 µm, 4.6 x 250 mm), Mobile phase: A: Bis-Tris Propane, pH 7.0, B: A + 1 M NaCl, pH 7.0,Flow rate: 0.8 mL/min, 170 bar; Detector: UV 280 nm, Column temperature: 25 ℃,Samples: 50, 150 mg each, PEG 10 kD conjugated BSA

min0 5 10 15 20 25 30 35

mAU

5

10

15

20

25

30

35

40

150 mg

50 mg

15

PG1001


Proteomix® SAX – 20 kD Pegylated BSA LoadingColumn: Proteomix® SAX-NP5 ( 5 µm, 4.6 x 250 mm), Mobile phase: A: Bis-Tris Propane, pH 7.0, B: A + 1 M NaCl, pH 7.0,Flow rate: 0.8 mL/min, 170 bar; Detector: UV 280 nm, Column temperature: 25 ℃,Samples: 50, 150 mg each, PEG 20 kD conjugated BSA

min0 5 10 15 20 25 30 35

mAU

0

5

10

15

20

25

30

35

150 mg

50 mg

16

PG1001


Proteomix® SAX – 30 kD Pegylated BSA LoadingColumn: Proteomix® SAX-NP5 ( 5 µm, 4.6 x 250 mm), Mobile phase: A: Bis-Tris Propane, pH 7.0, B: A + 1 M NaCl, pH 7.0,Flow rate: 0.8 mL/min, 170 bar; Detector: UV 280 nm, Column temperature: 25 ℃,Samples: 50, 100, 150 mg each, PEG 30 kD conjugated BSA

min0 5 10 15 20 25 30 35

mAU

0

5

10

15

20

25

30

35

40

150 mg100 mg50 mg

1

2

17

PG1001

SAX collected fractions on Zenix SEC-300peak 1 and 2 from previous slide

Column: ZenixTM SEC-300, ( 3 µm, 300 Å, 7.8 x 300 mm) Mobile phase: 150 mM Phosphate buffer, pH 7.0;Flow rate: 1 mL/min; Detector: UV 280 nm; Column temperature: 25 ℃;Samples: 30 kD pegylated BSA fractionated on SAX, fraction 1 and 2 concentrated with a 3kD Amicon Ultra

min2 4 6 8 10 12

mAU

0

10

20

30

40

50

60

70

Fraction 1Fraction 230 kD PEG-BSA mixture

18 www.sepax-tech.comBetter Surface Chemistry for Better Separation © Sepax Technologies, Inc.

PG1001

Documents

BSA pegylation - Sepax Technologies, Inc. · 2020. 10. 20. · BSA pegylation •BSA 5mg/mL, pI= 4.7, Sigma A7096, 65-70% free sulfyhydryl, cysteine 34 of BSA is not paired with another