Anion Exchange Column Regeneration

Regeneration

The following procedure should be satisfactory to remove common contaminants:
1. Wash with 2 column volumes of 2 M NaCl at 0.2 ml/min.
2. Wash with 4 column volumes of 1 M NaOH at 0.2 ml/min.
3. Wash with 2 column volumes of 2 M NaCl at 0.2 ml/min.
4. Rinse with at least 2 column volumes of distilled water at 0.2 ml/min until the UV-baseline and elutent pH are stable.
5. Wash with at least 4 column volumes of start buffer or storage buffer at 0.2 ml/min until pH and conductivity values have reached the required values.

If back pressure remains high, change top filter.

Save time by monitoring any cleaning procedure to check for elution of contaminants.  Depending on the nature of the contaminants, the following cleaning solutions can also be used: 100% isopropanol, 20% acetonitrile, 2 M NaOH, 75% acetic acid, 20% ethanol, 100% methanol or up to 6 M guanidine hydrochloride, cationic or non-ionic detergents.

Always rinse with at least 2 column volumes of distilled water after using any of these cleaning solutions. When using organic solvents, wash the column using sawtooth gradients e.g. run from 0–100% solvent in 5 column volumes then from 100–0% in 5 column volumes, including 1% trifluoroacetic acid in the water and organic solvent. If column performance is still not restored, inject a solution of 1 mg/ml pepsin in 0.1 M acetic acid containing 0.5 M NaCl and leave overnight at room temperature or one hour at 37 oC.

Depending on the contaminant, other enzymes may also be used, e.g. DNase. After any enzymatic treatment, repeat the steps to remove severe contamination described previously.

Resolubilization of protein precipitates

Many proteins are easily resolubilized in a small amount of the buffer to be used in the next chromatographic step. However, a denaturing agent may be required for less soluble proteins. Specific conditions will depend upon the specific protein. These agents must always be removed to allow complete refolding of the protein and to maximize recovery of mass and activity. A chromatographic step often removes a denaturant during purification. Table 17 gives examples of common denaturing agents.

Chemical stability

For daily use, MiniBeads are stable in all common aqueous buffers in the range pH 3–11 and in the presence of additives such as denaturing agents (8 M urea or 6 M guanidine hydrochloride), non-ionic or ionic detergents and up to 30% acetonitrile in aqueous buffers. Note that aqueous solutions of urea, ethylene glycol and similar compounds will increase the back-pressure due to increased viscosity. MiniBeads can be used with organic solutions such as dimethylsulfoxide, dimethylformamide or formic acid, but the separation properties of the media will change. Avoid anionic detergents with Mini Q. Avoid cationic detergents with Mini S. Avoid oxidizing agents.

Storage

For column storage, wash with 4 column volumes of distilled water followed by 4 column volumes of 20% ethanol. Degas the ethanol/water mixture thoroughly and apply at a low flow rate to avoid over-pressuring the column. Store at room temperature or, for long periods, store at +4° C to +8° C. Whenever possible, use the storage and shipping device if supplied by the manufacturer. Ensure that the column is sealed well to avoid drying out. Do not
freeze