HiTrap™ IEX Selection Kit from GE Healthcare

I recently received the HiTrap™ IEX Selection Kit from GE Healthcare. My project involves purification of a protein using conventional chromatography. The starting material includes other proteins with similar characteristics. However, these proteins differ in their iso-electric point, suggesting that the use of ion-exchange chromatography will prove successful.

To start this project, I thought it was important to run small pilot experiments to determine the chromatography profiles resulting from different ion exchange chromatography (IEX) columns. To that end, I searched the web for a selection kit that included several different anion and cation exchange columns and importantly, the column ideally should be small (1 ml) and pre-packed. This set up allows for quickly run, small scale chromatography and saves laborious time required for column preparation.

HiTrap™ IEX Selection Kit description: As stated on GE Healthcare’s website, “The kit allows fast and easy selection with seven different ion-exchange ligands on Sepharose™ Fast Flow and Sepharose™ XL, prepacked in 1 ml HiTrap™ columns.” This arrangement of Sepharose™ affords the use of a variety of different chromatography systems, both low and medium pressure, but if such a system is not available, the experiments can be done using a syringe. The small size of the prepacked columns means that pilot experiments can be done in a short period of time. The list of columns cover strong and weak ion exchangers and include the following matrixes: 1) Q Sepharose™ Fast Flow; 2) SP Sepharose™ Fast Flow; 3) CM Sepharose™ Fast Flow; 4) DEAE Sepharose™ Fast Flow; 5) ANX Sepharose™ 4 Fast Flow (high sub); 6) Q Sepharose™ XL; 7) SP Sepharose™ XL. The columns are supplied with detailed protocols for use. The columns can handle a flow rate of 4 ml/min, but it is recommended to run them at 1 ml/min for better separation. The pressure limit for all columns is 3 bar or 0.3 MPa or 43 psi. These columns can be used for small scale purification as well as a fast method for the development of a purification scheme.

The condition of the sample is very important in achieving the most effective separations. Ideally, samples should be in the same conditions as the start buffer. When working with small volumes during screening and scouting, it may be sufficient to dilute the sample in start buffer in order to lower the ionic strength and adjust the pH to a value similar to that of the start buffer.

In approximately two months, I ran 12 protein chromatography experiments from which we could conclude which direction to take with regard to the choice of column, desired pH, optimization of elution by salt (NaCl), flow-rate, sample preparation and also conditions for a step-wise elution.

The big advantage of the kit is that it allows for excellent optimization studies. Buffer composition, pH, flow rate, sample loading, and elution scheme can be optimized with small sample quantities prior to scale-up.

The only negative that I personally experienced are first, it happened that by using the AKTA system, I got air into the column and once this happened it was impossible to regenerate the column and the efficiency of separation dropped dramatically. Second, because of the small column volume and the low pressure these columns accommodate, sharp and complete separation was not easy and required a good amount of optimization of the parameters mentioned above.