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How to combine purification steps
The purification of crude biological samples can be divided into three distinct phases (Fig 3.1).
Fig 3.1. The three-stage strategy applied when purifying crude samples.
The capture (C) step aims at concentrating the sample and removing the bulk of the contaminants. Emphasis is primarily on speed and load capacity. Media with high load capacities and good flow properties are used often under step elution conditions.
The intermediate purification (iP) step(s) aims at separating the components of the now concentrated partially purified sample. Emphasis here is on maximum resolution since the remaining contaminants have rather closely related chromatographic properties. This is done by combining techniques of independent selectivities run in high resolution mode (gradient elution, smaller bead sizes etc.).
Flow rates and load capacities have to be restricted or resolution will suffer.
The polishing (P) step(s) serves to achieve the final purity and to eliminate contaminants such as polymers etc. and is also often used to condition the final product, e.g. to remove salts, when the final product is to be lyophilised.
Please look at the CiPP animation to get a clear understanding of the three-stage purification strategy. The animation requires that Macromedia Shockwave player is installed in your computer.
No chromatographic technique will provide 100% yield of active material and the overall yield will therefore depend on the number of steps included in the purification protocol.
As seen in figure 3.2, even a reasonable yield of 80% per step, results in only 20% overall yield after 8 purification steps.
Fig 3.2. Overall yields at different levels of yield/step.
Minimizing the number of steps (including inter-step conditioning) is therefore important to arrive at a high
overall yield.
To minimize the number of steps, method development should aim at optimising each step for the intended purpose and arranging them in an order that minimises inter-step treatments (Fig 3.3)
Fig 3.3 Start conditions and end conditions for commonly
used chromatography techniques.
Purification by removing the target molecule from the contaminants.
Affinity chromatography techniques are very specific for the target molecule or for a group of molecules with closely related biological properties. This makes them capable of
“fishing out” the target molecule (or the group), leaving all contaminants behind.
When applicable these techniques are to be preferred, since they drastically simplify the purification protocol.
Purification by removing the contaminants from the target molecule.
When a suitable affinity chromatography technique is not at hand, one has to rely on a sequence of general chromatography techniques to remove the contaminants.
A typical purification protocol when nothing is known about the target protein employs the IEX- HIC- GF sequence of purification steps.
The different techniques used in purification are not suitable for all stages of any given three phase strategy. Guidence on selection of appropriate three-phase strategies is given in Figures 3.4 to 3.7.
Fig 3.4 Strategies for low soluble proteins
Fig 3.5. Strategies for very dilute samples.
Fig 3.6. Strategies for crude samples.
Fig 3.7. Suitability ranking of chromatography techniques
with respect to the three-phase strategy. |
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