Affinity Chromatography - Department of Molecular and Cellular ...

0.6
0.4
0.2
0
A280
pH selected for elution
in a step gradient
pH
pH
7.0
6.0
5.0
4.0
Sample:
Column:
Binding buffer:
Elution buffer:
Flow:
System:
Cell culture supernatant containing
monoclonal IgG 1 , 90 ml
HiTrap rProtein A FF, 1 ml
100 mM sodium phosphate,
100 mM sodium citrate,
2.5 M sodium chloride, pH 7.4
100 mM sodium phosphate,
100 mM sodium citrate,
pH-gradient from 7.4 to 3.0
1 ml/min
ÄKTAFPLC
A 280 nm
3.0
150 200 250
ml
Fig. 8. Scouting for optimal elution pH of a monoclonal IgG 1
from HiTrap rProtein A FF, using a pH gradient.
Flow rates
It is not possible to specify a single optimal flow rate in affinity chromatography because
dissociation rates of ligand/target molecule interactions vary widely.
For ready to use affinity media follow the manufacturer's instructions and optimize further
if required:
-determine the optimal flow rate to achieve efficient binding
-determine the optimal flow rate for elution to maximize recovery
-determine the maximum flow rate for column re-equilibration to minimize total run times
To obtain sharp elution curves and maximum recovery with minimum dilution of separated
molecules, use the lowest acceptable flow rate.
Analysis of results and further steps
The analysis of results from the first separation can indicate if the purification needs to be
improved to increase the yield, achieve higher purity, speed up the separation or increase
the amount of sample that can be processed in a single run. Commonly used assays are
outlined in Appendix 8.
It is generally recommended to follow any affinity step with a second technique, such as a
high resolution gel filtration to remove any aggregates, or ligands that may have leached
from the medium. For example, Superdex can be used to separate molecules, according
to differences in size, and to transfer the sample into storage buffer, removing excess salt and
other small molecules. The chromatogram will also give an indication of the homogeneity
of the purified sample.
Alternatively, a desalting column that gives low resolution, but high sample capacity, can be
used to quickly transfer the sample into storage buffer and remove excess salt (see page 134).
Equipment selection
Appendix 2 provides a guide to the selection of purification systems.
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