Table 9.
Conditions for optimized pilot-scale two-step chromatography setup for Fab purification, along with tabulated details of the buffers, flow rate(s), number of CVs used for the purification of Fab molecule.
| Affinity Chromatography (AxiChrom 70/300 packed with Protein L resin) | |||
|---|---|---|---|
| Stage | Content | Flow rate (mL/min) | Number of CV |
| Equilibration | 20 mM sodium phosphate pH 7.6 | 230 | 5 |
| Load | Not adjusted | 100 | Variable |
| Wash | 20 mM sodium phosphate, pH 6.0 | 230 | 6 |
| Elution | 100 mM glycine-HCl, pH 2.5 | 230 | 10 |
| CIP | 0.1 M NaOH | 100 | 3 |
| Storage |
20 % ethanol |
230 |
3 |
| Cation Exchange Chromatography (AxiChrom 50/300 packed with SP-FF resin) | |||
| Stage | Content | Flow rate (mL/min) | Number of CV |
| Equilibration | 20 mM sodium phosphate pH 5.0 | 100 | 5 |
| Load | pH adjusted to 5.0 | 65 | Variable |
| Wash | 20 mM sodium phosphate 5.0 | 100 | 3 |
| Elution (linear gradient) | 20 mM sodium phosphate pH 8.5 | 100 | 30 |
| CIP | 0.5 M NaOH | 65 | 3 |
| Storage | 20 % ethanol | 100 | 3 |