Hydrophobic Interacting Chromatography requires
hydrophobic moieties on the outer portions of a protein and separates
the protein from a feed by its tendency to associate with other hydrophobic
molecules, such as phenyl or benzyne, in aqueous solutions. This tendency
is driven by the properties of water and the lower free energy of
the system achieved by minimized hydration shells. When a hydrophobic
molecule is immobilized to a solid support with a hydrophilic backbone,
such as agarose, great separations can be achieved by a reverse step
gradient elution, reducing the ionic strength of the elution buffer
over time. The higher salt concentrations promote the hydrophobic
interaction between the immobilized phenyl and the hydrophobic outer
portions of the protein by decreasing the entropy, leaving hydrophobic
association a prime candidate for reducing the entropy of the system
(reducing the order of water molecules). As the salt is removed from
the system gradually, the differences in hydrophobicity account for
the resolution and separation of different proteins.
Sterogene’s Phenyl Cellthru BigBead utilizes these principles
to directly capture the protein of interest from crude media, such
as cell culture. This can eliminate the requirement for filtration
or centrifugation, combining recovery and purification into one step.
It also minimizes the contact time of a desired protein with possible
degradative proteins, such as proteases and glycosidases. It use in
a standard, packed bed column is also highly desired as many early
attempts at direct capture have been limiting in that there is special
equipment to buy and limited flow rates. While most columns are prefitted
with a 10µm screen, Sterogene supples a 180µm screen to retrofit the
column, which allows the particulate material to pass through without
clogging the column. Once this is done, direct capture is an excellent
way to start off a recovery and purification. |
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