45
HIC
Five different hydrophobic surfaces and
selectivities
Tosoh Bioscience offers five HIC ligands featuring different
degrees of hydrophobicity and selectivity. The hydropho-
bicity of TOYOPEARLHIC resins increases through the ligand
series: Ether, PPG (polypropyleneglycol), Phenyl, Butyl, and
Hexyl (Figure 2).
Coordinating the hydrophobicity of the therapeutic target
to the resin hydrophobicity is critical for the best overall
purification performance. Too hydrophobic a resin for a
given protein can result in its irreversible binding to the
resin or a loss of enzymatic activity. Table I and II show
typical mass recovery and biological activity recovery data
for TOYOPEARL HIC resins.
An optimum HIC process step will balance high dynamic
binding capacity, adequate selectivity, good mass
recovery and retention of biological activity. The wide
range of TOYOPEARL selectivities enables a developer
to optimize protein separations at the extremes of the
hydrophobic spectrum. Highly retentive TOYOPEARL
Hexyl-type and TOYOPEARL Butyl-type resins are used
to separate hydrophilic proteins. These two resins should
also be considered for separations requiring a low salt
environment. TOYOPEARL Ether-type resin is used for the
purification of very hydrophobic targets such as certain
monoclonal antibodies and membrane proteins. These
proteins may bind irreversably to other more hydrophobic
resins. TOYOPEARL PPG-type and TOYOPEARL Phenyl-
type phases complement the other HIC ligands available in
the TOYOPEARL series and offer alternatives for mid-range
hydrophobic proteins.
hydrophobic interaction
chromatography
HIC ligand candidates
Ether
Phenyl
Butyl
O
OH
O
n
PPG
Hydrophobic
Hydrophilic
(OCH
2
CH
2
)
n
OH
OCH
2
CH
2
CH
2
CH
3
Hexyl
OCH
2
CH
2
CH
2
CH
2
CH
2
CH
3
Hic ligand candidates
figure 2
TOYOPEARL HIC resin
Ether-650M Phenyl-650M Butyl-650M
Bovine serum
84
62
76*
albumin
a
-chymotrypsinogen 96
88*
90
Cytochrome C
-
81*
87*
IgG
91
-
-
a
-Lactalbumin
90
-
-
Lysozyme
94
92
85
Ovalbumin
83
88
73
Ribonuclease A
-
72*
82*
Procedure: A 200 mL sample containing 200 mg of protein was loaded onto
a 7.5 mm column and eluted with a 60 minute gradient of 1.8 mol/L (*1.5
mol/L) to 0.0 mol/L ammonium sulfate in 0.1 mol/L sodium phosphate (pH
7.0). The mass recovery was determined spectrophotometrically at UV 280
nm and 25°C.
TABLE I
High mass recovery (%) of proteins
TABLe II
TOYOPEARL Protein
% Activity
HIC resin
recovery
Phenyl-650 Phytochrome
79
Butyl-650
Halophilic protease
85
Butyl-650
Poly (3-hydroxybutyrate) depolymerase
88
Butyl-650
Aculeacin-A acylase
82
Butyl-650
Opine dehydrogenase
81
Recovery of enzymatic activity of proteins
