亚粒径阳离子交换色谱(2)

Column Lifetime for Antibodix ? WCX 4.6 x 50 mm

Figure 11. Lifetime test for Antibodix ? WCX NP1.7 4.6 x 50 mm with a 4 x 10 mm guard column. Mobile phase A: 20 mM sodium phosphate buffer pH 6.5 and B: A + 1.0 M NaCl. The gradient was 10-100% B in 25 minutes with a 15 minute prewash. Flow rate was 0.3 mL/min. UV detection was set at 214 nm. 3 µL of sample was injected and the sample is a mixture of Aprotinin, Lysozyme and Ribonuclease A (1 mg/mL

each).

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9 MAb separation on Proteomix ® SCX NP1.7 4.6 x 100 mm vs. NP5 4.6 x 250 mm

Figure 12. Particle size and column length comparison for Proteomix ® SCX NP. Mobile phase A: 2.4 mM Tris, 1.5 mM Imidazole, 11.6 mM piperazine pH 6.0 and B: A + 0.5 M NaCl pH 10.5. The gradient was 0-100% B in 25 minutes with a 15 minute prewash. Flow rate was 0.75 mL/min (NP1.7) and 0.8 mL/min (NP5). UV detection was set at 280 nm. 5 µL of MAb 321 was injected on each column (1 mg/mL).

MAb separation on Proteomix ® SCX NP1.7 vs. Proteomix ® SCX NP5 (4.6 x 100 mm)

Figure 13. Particle size comparison for Proteomix ® SCX NP1.7 4.6 x 100 mm to Proteomix ® SCX NP5 4.6 x 100 mm. Mobile phase A: 2.4 mM Tris, 1.5 mM Imidazole, 11.6 mM piperazine pH 6.0 and B: A + 0.5 M NaCl pH 10.5. The gradient was 0-100%

B in 25 minutes with a 15 minute prewash. Flow rate was 0.75 mL/min. UV detection was set at 280 nm. 5 µL of MAb 321 was injected on each column (5

mg/mL).

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10 Separation of BSA Digest on Proteomix ® SCX NP1.7 2.1 x 30 mm

Figure 14. Separation of BSA tryptic digest on Proteomix ® SCX NP1.7 2.1 x 30 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 0.35 mL/min. UV detection was set at 214 nm. 5 and 10 µg of trypsin digested BSA was injected on the column.

Separation of Peptides on Proteomix ® SCX NP1.7 2.1 x 30 mm

Figure 15. Separation of four Peptides on Proteomix ® SCX NP1.7 2.1 x 30 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 0.35 mL/min. UV detection was set at 214 nm. 2 µL of the peptide

mixture was injected.

2 1

3

4 5

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11 Gradient Optimization for Peptide Separation on Proteomix ® SCX NP1.7

Figure 16. Gradient optimization for the separation of four Peptides on Proteomix ® SCX NP1.7 2.1 x 30 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 0.35 mL/min. UV detection was set at 214 nm. 2 µL of the peptide mixture was injected.

Particle Size Comparison for the Separation on Peptides on Proteomix ® SCX

Figure 17. Particle size comparison for Proteomix ® SCX NP1.7 2.1 x 30 mm to Proteomix ® SCX NP5 4.6 x 100 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 1.0 mL/min (for 5 µm) and 0.35 mL/min (for 1.7 µm). UV detection was set at 214 nm. 2 µL of the peptide mixture was injected.

2 1

3

4 5

2

1 3 4 5

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12 Shorter Run Time for Peptide Separation on Proteomix ® SCX NP1.7

Figure 18. Short run time and high resolution for the separation of five Peptides on Proteomix ® SCX NP1.7 2.1 x 30 mm compared to Proteomix ® SCX NP5 4.6 x 100 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 1.0 mL/min (for 5 µm) and 0.6 mL/min (for 1.7 µm). UV detection was set at 214 nm. 2 µL of the peptide mixture was injected.

Zoomed View of Short Run for Peptide Separation on Proteomix ® SCX NP1.7

Figure 19. Zoomed view of short run time and high resolution for the separation of five Peptides on Proteomix ® SCX NP1.7 2.1 x 30 mm compared to Proteomix ® SCX NP5 4.6 x 100 mm. Mobile phase A: 10 mM phosphate buffer pH 2.5, B: A + 1 M NaCl and C: 100% acetonitrile. Flow rate was 1.0 mL/min (for 5 µm) and 0.6 mL/min (for 1.7 µm). UV detection was set at 214 nm. 2 µL of the peptide mixture was injected.

2

1 3 4 5

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13 Analysis of Fab and Fc Fragments on Proteomix ® WCX NP1.7

Figure 20. Analysis of papain digested MAb fragments, Fab and Fc, on Proteomix ® WCX NP1.7 4.6 x 30 mm. Mobile phase A: 20 mM Sodium Acetate pH 5.15, B: A + 1 M LiCl. Flow rate was 1.5 mL/min. UV detection was set at 280 nm. 25 µL of the papain digested MAb 321 (1 mg/mL) was injected. Digestion condition: 5.0 mM

L-Cysteine, 2.0 mM EDTA and 0.1M Tris-HCl pH 7.6 (Papain: MAb=100:1). Incubate at 37 °

C for 3.5 hours, add 5% TFA to stop the reaction and chill on ice for 15 minutes.

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