Affinity Chromatography - Department of Molecular and Cellular ...

Applying CIPP
Imagine the purification has three phases: Capture, Intermediate Purification and Polishing.
Assign a specific objective to each step within the purification process.
The purification problem associated with a particular step will depend greatly upon the
properties of the starting material. Thus, the objective of a purification step will vary
according to its position in the process.
As shown in Figure 72, an important first step for any purification is correct sample
preparation and this is covered in more detail in Appendix 1.
In the capture phase the objectives are to isolate, concentrate and stabilize the target product.
The product should be concentrated and transferred to an environment that will conserve
potency/activity.
During the intermediate purification phase the objectives are to remove most of the bulk
impurities, such as other proteins and nucleic acids, endotoxins and viruses.
In the polishing phase most impurities have already been removed except for trace amounts
or closely related substances. The objective is to achieve final purity by removing any
remaining trace impurities or closely related substances.
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
Proteins are purified using purification techniques that separate according to differences in
specific properties, as shown in Table 11.
Table 11. Protein properties used during purification.
Protein property
Biorecognition (ligand specificity)
Charge
Size
Hydrophobicity
Technique*
Affinity (AC)
Ion exchange (IEX)
Gel filtration (GF)
Hydrophobic interaction (HIC), Reversed phase (RPC)
*Expanded bed adsorption is a technique used for large-scale purification. Proteins can be purified from crude sample
without the need for separate clarification, concentration and initial purification to remove particulate matter. The
STREAMLINE adsorbents, used for expanded bed adsorption, capture the target molecules using the same principles
as affinity, ion exchange or hydrophobic interaction chromatography.
Resolution
Speed
Recovery
Capacity
Every chromatographic technique offers a balance between resolution, capacity, speed and recovery.
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