Condition Screening for Scaling up using HiScreen IMAC FF

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

HiScreen IMAC FF is a ready-to-use column for purification of proteins by IMAC. The column is prepacked with 4.7 ml of uncharged IMAC Sepharose 6 Fast Flow. The high flow rate properties of this chromatography medium make HiScreen IMAC FF columns well suited for establishing optimal chromatographic conditions for scaling up as well as for small-scale purifications.

The medium can easily be charged with Ni2+, Co2+, Zn2+, Cu2+, Fe3+ or other metal ions. The columns are made of biocompatible polypropylene that does not interact with biomolecules. See Appendix 1 (Characteristics of Ni Sepharose®, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products) for the main characteristics of HiScreen IMAC FF.

The columns are used in an optimal manner with liquid chromatography systems such as ÄKTA.

HiScreen IMAC FF column

Fig 3.51. HiScreen IMAC FF column for establishing optimal chromatographic conditions for scaling up.

 

Sample and buffer preparation

Refer to Purification using IMAC Sepharose 6 Fast Flow for a general procedure for sample and buffer preparation.

Charging the chromatography medium with metal ions

  1. Prepare a 0.1 M solution of the desired metal ion (e.g., Cu2+, Zn2+, Co2+, Fe3+, or Ni2+) in distilled water. Salts of chlorides, sulfates, etc., can be used (e.g., 0.1 M CuSO4 or 0.1 M NiSO4).

Solutions of Zn2+ ions should have a pH of approximately 5.5 or lower to avoid solubility problems that arise at pH 6 or higher. Fe3+ ions should be immobilized at low pH (approximately pH 3.0) to avoid formation of insoluble ferric compounds.

  1. Wash the column with 25 ml of distilled water.
  2. Apply at least 2.5 ml of the metal ion solution to the column.
  3. Wash with 25 ml of distilled water and 25 ml of binding buffer (washing with binding buffer—to adjust pH—should be done even if the metal-charged column is only to be stored in 20% ethanol).

Wash only with water, not buffer, prior to applying the metal ion solution, otherwise unwanted precipitation may occur.

In some cases, a blank run may be needed (see below).

The column does not have to be stripped and recharged between each purification if the same protein is going to be purified; it may be sufficient to strip and recharge it after approximately five purifications, depending on the sample properties, sample volumes, metal ion, etc.

 

Optional blank run:

Perform a blank run without reducing agents before applying buffers/samples containing reducing agents. Likewise, a blank run is recommended for critical purifications where metal ion leakage during purification must be minimized.

Use binding buffer and elution buffer without reducing agents.

  1. If the column has been stored in 20% ethanol after metal ion charging, wash it with 25 ml of distilled water.
  2. Wash with 25 ml of the buffer that has been chosen for protein elution, for example, imidazole elution buffer or low-pH elution buffer.
  3. Equilibrate with 25 to 50 ml of binding buffer. Imidazole equilibration can be monitored by absorbance, e.g., at 220 nm.

 
Purification

The recommended flow rate for HiScreen IMAC FF is 2.3 ml/min (300 cm/h flow velocity).

  1. After the column preparation (charging with metal ions), equilibrate with at least 5 column volumes of binding buffer. Avoid introducing air into the column.

To prevent leakage, ensure that the connectors are tight. Use fingertight 1/16" connector.

In some cases, we recommend a blank run before final equilibration/sample application (see above).

  1. Adjust the sample to the chosen starting conditions and load on the column.
  2. Wash with 5 to 10 column volumes of binding buffer until the UV trace of the effluent returns to near baseline.
  3. Elute either by linear gradient elution or step elution at recommended flow rates. If required, the collected eluted fractions can be buffer exchanged or desalted.
    Linear gradient elution
    : Elute with 0% to 100% elution buffer (up to 500 mM imidazole) in 10 to 20 column volumes.
    Step elution
    : Elute with 5 column volumes of elution buffer including imidazole at chosen concentration. Repeat at higher imidazole concentrations until the target protein has been eluted.
  4. If required, strip the column from metal ions and perform CIP to clean the column.
  5. Re-equilibrate the column with 5 to 10 column volumes of binding buffer or until the UV baseline, eluent pH, and conductivity reach the required values.

Do not exceed the maximum recommended flow and/or back pressure for the column.

For A280 measurements, use the elution buffers as blanks. If imidazole needs to be removed, use a desalting column (see Chapter 11, Desalting/buffer exchange and concentration). Low-quality imidazole will give a significant background absorbance at 280 nm.

Materials

     
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