IEX technique is very common in biopharmaceutical production due to the robustness,
reliability and scaleability.
In ion exchange chromatography, charged substances are separated via column materials
that carry an opposite charge.
The amphoteric nature of proteins, which are multivalent
ions, makes the method useful. Depending on the pH of their environment,
proteins are anions or cations. The protein's net charge is neutral
only at the iso-electric point (pI). At pH values above the pI, the
protein is negatively charged, at pH values below pI, it is positively
charged. Therefore, it is important to operate at pH values where
the exchangers are mostly ionized and the biopolymers are charged,
i.e. about 0.5-1.0 unit away from the pI. The desorption of the proteins
from the column is achieved by increasing salt concentrations or pH
changes when the protein loses charge. Substances that have a higher
charge density are bound correspondingly stronger to the column while
the others elute rapidly.
Commonly Used Functional Groups
Two exchanger types are available:
Cation exchangers which are negatively charged (acidic) for the binding of
positively charged proteins and positively charged anion exchangers (basic) which
bind negatively charged proteins. They can be divided into those with weakly basic
or acidic character or strongly basic or acidic character. With strongly basic or
acidic materials all functional groups are always present in ionized form (within
their specified operating range) independent from the pH value.
For example, the quaternary amino groups (TMAE) of strong anion exchangers are
positively charged, while the sulfonic acid groups (SO3-) of strong cation exchangers
are negatively loaded.
The weakly basic types consist of secondary and tertiary amino functional groups;
the weakly acidic types of carboxyl functional group. Thus weakly basic exchanger
should only be used at pH values under 8.5, weakly acidic exchangers only at pH values
above 6. Outside these ranges strongly basic, or strongly acidic exchangers should be used.
Getting Started Now (Practical Hints)
Any of the conventional buffers can be used for ion exchange chromatography. However,
positively charged buffering ions should be used on anion exchangers to avoid an
interaction or binding to the functional group. Therefore, Tris (pKa 8.2) is preferred
with Cl- as counterion. For cation exchangers, the buffering ion should be negatively
charged, for example carbonate, acetate or MES, and the counterion K+ or Na+.
Phosphate buffers are generally used on both exchanger types.
As the starting condition the highest salt concentration should be used that
permits binding of the target protein. The final chromatographic condition should
be the lowest ionic strengths which allows the elution of the protein of interest.
A third and higher concentration of salts in the buffer can be recommended as a
washing step before the re-use of the column.
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If the pl of the protein is known, select Fractogel® EMD TMAE or DEAE at a pH of
the buffer about 1-2pH units above the pl. |
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If the protein doesn't bind, use Fractogel® EMD SO3 or COO-
at a pH about 1-2 pH units below the pl. |
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If the pl of the protein is unknown check Fractogel® EMD TMAE or DEAE at pH 8
because most of the proteins are acidic; if you have no success use Fractogel® EMD
SO3 or Fractogel® EMD COO- at pH 6. |
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Always check the stability of the target protein to avoid denaturation. |
Flow Rate:
Depending on the desired resolution, linear flow rates to 380cm/hr (= 5ml/ml for a 1cm column)
can be used. The protein binding capacity is not affected up to these flow rates.
Elution:
In order to achieve optimum results, the gradients used for elution should not be too steep.
A gradient volume which is 5-8 times more than the column volume should be sufficient.
Depending on the individual separation problem step gradients can also be used. As a
rule the gradients are generated by a Buffer A without salt and a Buffer B, containing
1M NaCl (or another salt).
Column Dimensions:
As a rule, short columns with a large diameter should be used.
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