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5. Cleaning, Sanitization and Storage
For best performance of coupled CNBr-activated Sepharose 4 Fast Flow over
a long working life, follow the general procedures described below. In all
cases, we recommend testing the procedures at small scale first.
Equilibration
After packing, and before a chromatographic run, equilibrate with working
buffer by washing with at least 5 bed volumes.
Cleaning-In-Place
Cleaning-in-place, (CIP), is a cleaning procedure which removes
contaminants such as lipids, precipitates or denatured proteins that may
remain in the packed column after regeneration. Such contamination is
especially likely when working with crude materials. Regular CIP prevents
the build-up of these contaminants in the packed bed, and helps to maintain
the capacity, flow properties and general performance of the medium.
A specific CIP protocol should be designed for each process according to the
type of contaminants present and stability of coupled ligand. The frequency
of CIP depends on the nature and the condition of the starting material and
other process requirements, but one CIP cycle is generally recommended
every 1–5 separation cycles. Following are generally recommended
procedures.
CIP protocol
Precipitated or Wash with 2 column volumes of
denatured 6 M guanidine hydrochloride. Wash
substances substances immediately with at least
5 column volumes of sterile filtered binding
buffer.
Hydrophobically Wash the column with 2 column volumes of
bound substances a non-ionic detergent (conc. 0.1–0.5%).
Wash immediately with at least 5 column
volumes of sterile filtered binding buffer.