Bio-Rad Media Sampler Pack - Manual (Page 10)

minimizes product loss due to overly strong product interaction with the media. If
the target molecule is in the flow-through, sodium citrate is often used at
concentrations of 500–600 mM for loading.
Sample Preparation
Adjust salt concentration and pH as necessary for desired binding of target or
contaminants. This is best done by mixing the correct amount of liquid
concentrate of salt into the sample load and then adjusting the pH.
Sample Load and Adsorption
The sample load is determined empirically by loading and evaluating breakthrough
of target or contaminants. Since HIC is an adsorption technique, the sample
volume is not a critical factor. Large volumes of very dilute feed such as cell
culture supernatant and clarified lysates can be loaded onto the media without
prior concentration. If the salt concentration of the sample load is lower than what
is used to equilibrate the column before loading, the protein of interest can show
low binding capacities, or can begin to elute from the column with unwanted
contaminants.
Wash through
After loading of the column, follow with 2 to 3 CV of the high ionic strength buffer.
This will wash out any unbound materials.
Elution
Elute target molecules with either a step or linear gradient. Usually, the salt
concentration at which the desired product binds is predetermined at small scale.
With this knowledge, the pH and salt concentration used in wash-through are
adjusted to eliminate the maximum amount of contamination before starting
elution of the target.
Regeneration
After each run, the packed bed should be washed with 3–5 CV of a low ionic
strength buffer such as 20 mM sodium citrate or 20 mM HEPES. This is followed
with 3–5 CV of water. If contamination remains, wash with 2–3 CV of 0.12 M
phosphoric acid in 1% acetic acid, pH 1.5. For very difficult cleaning, follow the
acid wash with a combination of nonionic detergents and then 30–70% ethanol.
Cleaning-in-Place (CIP)
If a column no longer yields reproducible results, the media may require a
thorough CIP to remove strongly bound contaminants. Acceptable CIP agents
include 25% acetic acid, 8 M urea, 1% Triton X-100, 70% ethanol, 30%
isopropyl alcohol, 1 N HCl, and 6 M guanidine-HCl. Cleaning with NaOH is not
recommended because the t-butyl and methyl groups are cleaved from the media
under strongly basic conditions. If it is absolutely necessary to use NaOH, use
0.1 N for 1 hr, and then wash immediately with a low pH, high-salt buffer such as
600 mM sodium citrate, pH 5.
8
LIT486C.qxd 11/2/2004 3:12 PM Page 8