HIC in a Purification Strategy (Cipp)

To ensure efficient, reproducible purification giving the required degree of purity, it is beneficial to develop a multi-step process using the purification strategy of Capture, Intermediate Purification and Polishing (Cipp), shown in Fig 49.

Strategy for protein purification

Fig 49. Strategy for protein purification.

Imagine the purification has three phases: Capture, Intermediate Purification and Polishing.

The optimal selection and combination of purification techniques for Capture, Intermediate Purification and Polishing is crucial for an efficient purification.

Selection and combination of purification techniques for Cipp

Proteins are purified using purification techniques that separate according to differences in specific properties, as shown in Table 15.

Table 15. Protein properties used during purification.

Protein property Technique
Size Gel filtration (GF)
Charge Ion exchange (IEX)
Hydrophobicity Hydrophobic interaction (HIC), Reversed phase (RPC)
Biorecognition (ligand specificity) Affinity (AC)

 Every technique offers a balance between resolution, capacity, speed and recovery

Fig 50. Every technique offers a balance between resolution, capacity, speed and recovery.

Select a technique to meet the objectives for the purification step.

Choose logical combinations of purification techniques based on the main benefits of the technique and the condition of the sample at the beginning or end of each step.

A guide to the suitability of each purification technique for the stages in Cipp is shown in Table 16.

Table 16. Suitability of purification techniques for Cipp

Technique Main features Capture Intermediate Polishing Sample start condition Sample end condition
IEX High capacity
High resolution
High speed
*** *** *** Low ionic strength
Sample volume not limiting
High ionic strength or pH change
Concentrated sample
HIC Good capacity
Good resolution
High speed
** *** * Low ionic strength
Sample volume not limiting
Low ionic strength or pH change
concentrated sample
AC High capacity
High resolution
High speed
*** *** ** Specific binding conditions
Sample volume not limiting
Specific elution conditions
concentrated sample
GF High resolution using Superdex® media   * *** Limited sample volume (<5%total column volume) and flow rate range Buffer exchanged (if required)
dilute sample
RPC High resolution   * *** Sample volume usually not limiting additives may be required In organic solvent, risk loss of biological activity

Minimize sample handling between purification steps by combining techniques to avoid the need for sample conditioning. The product should be eluted from the first column in conditions suitable for the start conditions of the next column (see Table 16).

Ammonium sulfate, often used for sample clarification and concentration (see Appendix 1), leaves the sample in a high-salt environment. Consequently HIC, which requires high salt to enhance binding to the media, becomes the ideal choice as the capture step. The salt concentration and the total sample volume will be significantly reduced after elution from the HIC column. Dilution of the fractionated sample or rapid buffer exchange using a desalting column will prepare it for the next IEX or AC step.

Gel filtration is a non-binding technique unaffected by buffer conditions, but with limited volume capacity. GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC) since the target protein will be eluted in a reduced volume and the components from the buffer will not affect the gel filtration process.

Selection of the final strategy will always depend upon specific sample properties and the required level of purification.

  • For any capture step, select the technique showing the most effective binding to the target protein while binding as few of the contaminants as possible, that is, the technique with the highest selectivity and/or capacity for the target protein.
  • A sample is purified using a combination of techniques and alternative selectivities. For example, in an IEX-HIC-GF strategy, the capture step selects according to differences in charge (IEX), the intermediate purification step according to differences in hydrophobicity (HIC) and the final polishing step according to differences in size (GF).
  • If nothing is known about the target protein use IEX-HIC-GF. This combination of techniques can be regarded as a standard protocol.
  • Consider the use of both anion and cation exchange chromatography to give different selectivities within the same purification strategy.

The objective of a capture step is to quickly bind the protein(s) of interest from the crude sample and isolate them from critical contaminants such as proteases and glycosidases. The target protein(s) are concentrated and transferred to an environment that will conserve potency/activity. Removal of other critical contaminants may also be achieved by careful optimization of binding conditions.

Focus is on capacity and speed in a capture step. It may be advisable to compromise on resolution in order to maximize the capacity and/or speed of the separation in this first step.

HIC media for capture steps should offer high speed and high capacity.

  1. Sepharose® Fast Flow (90 μm mean particle size) — good resolution at flows up to 300 cm/h.
  2. Use Sepharose® High Performance (34 μm particle size) HIC media for capture or scale-up when selectivity is satisfactory, high resolution is a priority and if lower flow rates (to compensate for a higher back pressure) are acceptable.
  3. Use SOURCE (15 μm mean particle size) if the required selectivity is not available in a medium of larger particle size.

Select start conditions that minimize binding of contaminants and so help to maximize the binding capacity for the target protein(s). This will facilitate a fast, simple step elution of the concentrated target protein(s).

Materials