Buffers for IMAC

Immobilized metal affinity chromatography steps are typically operated at close to neutral pH to ensure that the His residues that interact with the metal ions are uncharged and can form coordination linkages. Since the ligands on IMAC resins are negatively charged (such as IDA), buffers for this technique usually include a moderate salt concentration (0.2 to 0.5 M NaCl or equivalent) to prevent nonspecific ionic interactions with any uncharged sites. Common buffers employed for IMAC include phosphate and acetate with low concentrations of Tris buffer also being suitable on most resins.

Elution from IMAC resins can be effected by decreasing mobile-phase pH (which causes His residues to acquire a positive charge and cease to chelate metal ions) or by employing a mobile-phase modulator such as imidazole, which can also chelate with the metal ions. Harsher methods such as using EDTA to strip the metal ions, and thus elute protein associated with them, are not used in biopharmaceutical production, since a high concentration of metal ion would end up in the elution pool. While employing low pH elution, it should be ascertained if the ligand-Me2+ linkage is strong enough so that metal ions do not leach out. Step or linear gradient elution with imidazole is the most commonly used elution method. However, it must be recognized that imidazole itself binds quite strongly to the metal ions and hence does not function in quite the same way as salt concentration does in IEX. There can be a significant delay in breakthrough of a step or linear gradient from the column due to retention of imidazole on the resin. Imidazole breakthrough profiles can be monitored at 230 nm in the absence of a protein load. To achieve more reproducible elution profiles, the column is often presaturated with imidazole and a low concentration (0.5 to 2 mM) can also be included in the column load and wash buffers, if binding capacity does not suffer substantially.

Immobilized metal affinity chromatography media can tolerate chaotropes, organic solvents, and detergents quite well. These agents can be employed as wash buffer additives to selectively remove impurities. More typically however, the wash will consist of a buffer with either lower pH than the load or with a moderate concentration of imidazole in-between that of the load and elution conditions.

Ethylene diamine tetraacetic acid is often added to the elution pool following IMAC to chelate any metal ions that may have leached off the column during purification. EDTA is included in the strip buffer to remove metal ions from the column after each use. Following the metal ion strip, the column can be sanitized and stored in sodium hydroxide.

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