Chromatography Steps and Buffers to Prepare Resin for On-Resin Growth Experimentsa
| Chromatography Step | Buffer | pH | Column Volumes |
|---|---|---|---|
| Equilibration | 25 mM Tris + 100 mM NaCl | 7.5 | 2 |
| Load | HCCF | 7 | 25 |
| Wash | 25 mM Tris + 100 mM NaCl | 7.5 | ≤3 |
| Secondary Wash | 25 mM Tris + 1 M NaCl | 7.5 | 4 |
| Wash | 25 mM Tris + 100 mM NaCl | 7.5 | 3 |
| Elution | 0.1 M Acetic Acid | 2.9 | 4 |
| Equilibration | 25 mM Tris + 100 mM NaCl | 7.5 | 5 |
↵a Protein A chromatography media with silica (ProSep-vA Ultra) or agarose (MabSelect SuRe) backbones were cycled as indicated or used fresh to create “fouled” and “naive” resin conditions for mycoplasma spike.