IN 45 100 010 • AA • 6 / 12
Optimization
The following paragraphs will give indications on some parameters that can be tuned to find the optimal
conditions for the purification.
Buffer selection
Choosing a buffer with optimal binding and elution conditions for the target protein will improve the result of
the purification. The buffer should be selected to provide an optimal capacity and with a p
K
a
-value within 0.5
units from the intended pH. Table 3 shows examples of buffers that can be used for ion exchange
chromatography, however the buffer choice will be depending on the target molecule and aim of the
purification procedure. For other useful buffers and their p
K
a
-values at 25 °C see reference: Methods in
Enzymology, Volume 463, pp 46-47, Burgess, R.R and Deutcher M. P.
Table 3. Example of buffers for model protein purification using BabyBio S, BabyBio Q and BabyBio DEAE. Other buffers can possible be used.
Buffer
Product
Buffer composition
Binding buffer
BabyBio S
50 mM Na-phosphate, pH 7.0
Binding buffer
BabyBio Q
50 mM Tris-HCl, pH 7.4
Binding buffer
BabyBio DEAE
50 mM Tris-HCl, pH 7.4
Elution buffer
BabyBio S
50 mM Na-phosphate, 1 M NaCl, pH 7.0
Elution buffer
BabyBio Q
50 mM Tris-HCl, 1 M NaCl, pH 7.4
Elution buffer
BabyBio DEAE
50 mM Tris-HCl, 1 M NaCl, pH 7.4
Preferably, select buffer substances with opposite charge to the resin. A buffer substance that interacts with
the charged groups in the resin may cause local pH disturbances that disturbs the separation. Usually, low
conductivity in the binding buffer is preferred but optimization with regard to pH and conductivity can improve
binding capacity. An increase in ionic strength may decrease the ability of contaminants bind while the target
protein remains bound. However, chromatographic conditions selection should be such that the target protein
is stable during purification.
