HIC for Intermediate Purification

The objective of intermediate purification steps is to remove most of the significant impurities such as proteins, nucleic acids, endotoxins and viruses. In a typical intermediate purification step, speed is less critical since sample volume has been reduced and damaging contaminants have been removed during capture. Focus is on capacity and resolution in order to maintain productivity (amount of target protein processed per column in unit time) and to achieve as high selectivity (purity) as possible. Consequently, a gradient elution will usually be required.

  • Use a technique with a selectivity that is complementary to that used in the capture step.

Media for intermediate purification should offer high capacity and high resolution. Select as follows:

  1. Sepharose® High Performance (34 μm mean particle size) — for high resolution at flows up to 150 cm/h.
  2. Sepharose® Fast Flow (90 μm mean particle size) — for good resolution at flows up to 300 cm/h or when required selectivity is not available on a Sepharose® High Performance matrix.
  3. SOURCE 15 (15 μm mean particle size) — if the required selectivity is not available in a medium of larger particle size. Samples should be free from particulate matter.

Purification of Fab fragment

Figure 55 shows a classic protocol, IEX-HIC-GF, for purification of a Fab fragment against the gp 120 envelope of the HIV-1 virus. The fragment, which was expressed in the periplasmic space of E. coli, has a molecular mass of Mr 50 000 and isoelectric point (pI) ~11. The high pI made cation exchange the most suitable ion exchange step for the initial capture step. The high capacity and good resolution of Phenyl Sepharose® 6 Fast Flow (high sub) was well suited to intermediate purification, followed by a polishing step on gel filtration.


Starting Material: E. coli periplasmic expressed target protein
Capture: SP Sepharose® Fast Flow
Intermediate Purification: Phenyl Sepharose® 6 Fast Flow (high sub)
Polishing: Superdex® 75 Prep Grade

Purification of a Fab fragment

Fig 55. Purification of a Fab fragment.