51
HIC
Protein aggregate removal
The larger pore TOYOPEARL products such as TOYOPEARL
Butyl-650 and
TOYOPEARL Phenyl-650 resins are very
useful for protein aggregate
separation and removal. Early
work by Karger et al
1
in 1989 involving
proteins and aggre-
gates larger than 200 kDa demonstrated the effectiveness
of HIC for this application.
Glycoproteins
TOYOPEARLHICresinscanpurifyglycoproteins,whichoften
bind irreversibly to saccharide-based chromatographic
media. Figure 16 shows the purification of a large glyco-
protein on TOYOPEARL Butyl-650S resin.
DNA plasmid purification and endotoxin
removal
TOYOPEARL Hexyl-650C resin was successfully used for
plasmid DNA purification by Cambrex, Baltimore, MD (US
patent 6,953,686). Hexyl-650C was shown to be the most
effective among HIC resins for endotoxin removal with
capacities exceeding 2 million EU/mL of resin. Additionally,
RNA and protein impurities were effectively eliminated.
Hexyl-650C was also effective in separating the supercoiled
and open circular forms of plasmid DNA.
Removal of misfolded proteins
Because misfolded proteins will generally be more hydro-
phobic than the native protein, TOYOPEARL Butyl-650M
resin is used frequently for the removal of misfolded
proteins. In many cases flow through chromatography
can be accomplished under eluent conditions binding the
misfolds while allowing the target to flow through the
column.
Go to
to check
our database on the website for additional applications.
1
Grinberg, N. Blanco, R. Yarmush, D. Karger, B. L. Protein
Aggregation in High Performance Liquid Chromatography:
Hydrophobic Interaction Chromatography of ß-Lactoglobulin,
Anal. Chem. 1989, 61, 514-520.
hydrophobic interaction
chromatography
figure16
Large glycoprotein purified on Toyopearl Butyl-650S
Fraction Number (8 mL/fraction)
Absorbance, 280nm
Antibacterial activity (%-Inhibition)
2.0
1.0
0
20
10
30
70
100
Column:
Sample:
Elution:
Detector: UV @ 280nm
Toyopearl Butyl-650S, 22mm ID x 26cm
crude protein from sea hare
Aplysia kurodai
multi-step (NH
4
)
2
SO
4
in 50mmol/L phosphate buffer (pH 7.0)
A. load & wash: 40% saturated (NH
4
)
2
SO
4
B. 20% saturated (NH
4
)
2
SO
4
C. 0% saturated (NH
4
)
2
SO
4
40 50 60
80 90
A.
C.
B.
100
50
0
Column: TOYOPEARL Butyl-650S, 22 mm ID x 26 cm L
Sample: crude protein from sea hare Aplysia kurodai;
Mobile phase: multi-step (NH
4
)
2
SO
4
in 50 mmol/L phosphate buffer (pH 7.0);
A. load & wash: 4 % saturated (NH
4
)
2
SO
4
; B. 20 % saturated (NH
4
)
2
SO
4
;
C. 0% saturated (NH
4
)
2
SO
4
; Detector: UV @ 280 nm
Large glycoprotein purified on TOYOPEARL Butyl-650S
ORDERING INFORMATION
ToyoScreen RoboColumns columns for HIC:
Part # Product description
Package
0045031 ToyoScreen RoboColumn Phenyl-600M, 200 µL
0.2 mL x 8 columns
0045032 ToyoScreen RoboColumn Phenyl-600M, 600 µL
0.6 mL x 8 columns
0045033 ToyoScreen RoboColumn Butyl-600M, 200 µL
0.2 mL x 8 columns
0045034 ToyoScreen RoboColumn Butyl-600M, 600 µL
0.6 mL x 8 columns
0045035 ToyoScreen RoboColumn PPG-600M, 200 µL
0.2 mL x 8 columns
0045036 ToyoScreen RoboColumn PPG-600M, 600 µL
0.6 mL x 8 columns
0045037 ToyoScreen RoboColumn Phenyl-650M, 200 µL
0.2 mL x 8 columns
0045038 ToyoScreen RoboColumn Phenyl-650M, 600 µL
0.6 mL x 8 columns
