Introduction:

Ion exchange chromatography is used for separating proteins with different charge and this gives a very high resolution separation with high sample loading capacity. The separation of proteins is based on the reversible interactions between a charged chromatographic medium and with an oppositely charged protein molecule.

Principle and Procedure:

Charged proteins bind to the medium as they are being loaded on to a chromatographic column. Then the physiological conditions are changed in such a way that the bound proteins are eluted differentially. This protein elusion is done by increasing the salt concentration or by changing the pH. These changes are made gradually. Most commonly protein or samples are eluted with salt sodium chloride, using a gradient elution. Target protein or proteins of interest are collected in a purified, concentrated form.

The net surface charge of protein varies depending upon the surrounding pH of the medium. When pH of the surrounding medium is higher than its isoelectric point, then the protein will bind to an anion exchanger. When the surrounding Ph is below its isoelectric point then the protein will bind to a cation exchanger. Ion exchange chromatography can be repeated at different pH values, hence can be used to separate several different proteins depending upon their surface charges.

Choice of ion exchanger:

For most protein purification steps it is suggested to begin with a strong exchanger, this allows to work over a broad range of pH. If the isoelectric point of the required protein is below neutral pH (pH 7.0) or unknown, then strong anion exchanger are used to bind the target.

Sample volume and capacity:

Ion exchange chromatography technique is independent of sample volume provided, that the ionic strength of the sample is low and the target molecule (protein of interest) is highly charged. The total amount of loaded protein and which binds to the column should not exceed the total binding capacity of the chromatography column.

Media selection:

When selecting chromatographic medium parameters such as scale of purification, resolution required, speed of separation, sample stability and also media binding capacity should be taken into consideration.

Sample Preparation:

To ensure efficient binding of protein of interest during sample application, it should be maintained at the same pH and ionic strength as that of the starting buffer. Sample must be free of particulate matters before applying into the chromatographic colum.

Column Preparation:

Pre-packed columns are used since they increase the speed of separation and also they increase the efficiency of this technique. That is by using pre-packed columns we can get reproducible results along with high level of performance.

Buffer Preparation:

Selected medium and buffering ions should have the same surface charge. Concentration of the buffer should be sufficient to maintain buffering capacity and also the pH during sample application and also while increasing concentration of the salt.

When protein of interest have to be isolated from a sample of unknown charges, first should try all these conditions,

1. Anion Exchange

Gradient: 0-100% elution buffer B in 10 - 20 column volumes
Start buffer A: 20 mM Tris-HCl, pH 8.0
Elution buffer B: 20 mM Tris-HCl + 1 M NaCl, pH 8.0

2. Cation Exchange

Gradient: 0-100% elution buffer B in 10 - 20 column volumes
Start buffer A: 20 mM Na2HPO4.2H2O, pH 6.8
Elution buffer B: 20 mM Na2HPO4.2H2O + 1 M NaCl, pH 6.8

Method of Development:

1. Optimum ion exchanger is first selected by using small columns, this saves a lot of time and also sample. For example pre-packed HiTrap columns can be used.
2. Optimum pH is selected by beginning with pH unit away from 0.5-1 from the isoelectric point of the target protein or protein of interest.
3. Steepest gradient should be selected to get an acceptable resolution at the selected optimum pH.
4. Highest flow rate which maintains resolution is being selected and this helps in minimizing required separation time.
5. Step elusion method can also be followed in large scale separation of target protein.

Cleaning, sanitisation and sterilisation

Cleaning, sanitisation and sterilization method or procedures vary depending upon the type of sample and medium. Usually guidelines are provided with the medium and also with pre-packed columns.

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