Bio-Rad Bio-Scale™ Mini UNOsphere SUPrA™ Affinity Cartridges - Manual (Page 12)

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Section 6
Screening Conditions
Because different antibodies will have differing levels of affinity for UNOsphere
SUPrA™ media, it is highly recommended to assess the behavior of the target
antibody on the media. To do so, it is best to test for binding under conditions that
will bind the widest range of antibodies, followed by a linear elution protocol to
assess optimal elution conditions, before further refining the method.
It is important before undertaking this process to ensure that the target antibody is
stable and soluble under the full range of conditions used for the screening.
Using a BioScale™ Mini column (1 or 5 ml) packed with UNOsphere SUPrA media
A: 0.02 M sodium phosphate, 0.02 M sodium citrate, pH 7.5.
B: 0.02 M sodium citrate, 0.1 M sodium chloride, pH 3.
Equilibrate column the with 10 CV buffer A (see note below).
Inject a small sample of antibody either as is, or at 1:10 dilution in buffer A (see note
below).
Wash the loaded columns with buffer A, until effluent absorbance returns to
baseline.
Elute with a 10 CV linear gradient to buffer B; collect fractions.
Wash the column with 5 CV buffer B.
Note: Boric acid may be added to the binding/equilibration buffer to obtain a
higher pH range. Up to 1M sodium sulfate may be added to enhance binding.
Fig. 2. A simulated chromatography profile from which HETP and As values are
calculated.
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