IEX Column Phase Screening Service
| Sample: | Monoclonal antibody F, MW 145 kD, pI=8.4 |
| Goal: | In search of most suitable stationary phase and separation conditions for the analysis of antibody F variants. |
| Approach: | Screen variety of cation exchange stationary phases (Proteomix SCX, WCX and Antibodix WCX) at analytical level (4.6 × 250 mm) with mobile phase screening. |
| Conclusion: | Antibodix NP5 (5 µm, 4.6 × 250 mm) column with NaCl salt gradient at pH 7.5 offers the best selectivity and resolution for the antibody variant separation among the phases screened. |
LiCl salt gradient
Column: Proteomix SCX-NP5, WCX-NP5 and ABX WCX-NP5
Mobile phase: A: 20 mM NaAc, adjust pH to 5.15 by 380 µL HAc, B: A + 1 M LiCl
Flow rate: 0.8 mL/min
Detector: UV 280 nm
Column temperature: 30 °C
Injection volume: 10 µL
Pressure: 79 bar
NaCl salt gradient pH 7.5
Column: Proteomix SCX-NP5, WCX-NP5 and ABX WCX-NP5
Mobile phase: A: 20 mM Phosphate buffer, pH 7.5; B: A + 1 M NaCl
Flow rate: 0.8 mL/min
Detector: UV 280 nm
Column temperature: 30 °C
Injection volume: 10 µL
Pressure: 79 bar
pH/salt gradient
Column: Proteomix SCX-NP5, WCX-NP5 and ABX WCX-NP5
Mobile phase: A: 2.4 mM Tris, 1.5 mM Imidazole, 11.6 mM piperazine, adjust pH to 6.0 by 10 M NaOH; B: A + 0.5 M NaCl, adjust pH to 10.5 by 10 M NaOH
Flow rate: 0.8 mL/min
Detector: UV 280 nm
Column temperature: 30 °C
Injection volume: 10 µL
Pressure: 79 bar