48
HIC
Pore size optimization
Most TOYOPEARL HIC products are derived from the
versatile size exclusion resin, TOYOPEARL HW-65 (100 nm
mean pore size), as the base bead for the majority of
protein separations. But the pore size and accessible
surface area of TOYOPEARL resins can be optimized for a
given protein. More accessible surface area increases the
dynamic binding capacity (DBC) of the bead for a partic-
ular therapeutic target. This has led to the development of
two specialty lines of HIC materials with higher dynamic
binding capacities.
High dynamic binding capacity TOYOPEARL
process resins
For monoclonal antibodies a pore size of 75 nm s optimum.
A specially made base resin, TOYOPEARL HW-60, has
this pore size. Three ligands are available on TOYOPEARL
HW-60: polypropylene glycol (PPG), phenyl, and butyl.
A comparison of their DBCs with TOYOPEARL
Phenyl-
650M resin is shown in Figure 9. The selectivities of
TOYOPEARL
Butyl-600M, TOYOPEARL PPG-600M and the
TOYOPEARL Phenyl-600M resins, are shown in Figure 10.
hydrophobic interaction
chromatography
figure10
Comparison of Toyopearl 600M series resins
Column:
7.5cm ID X 7.5cm
Sample:
1 g/L RNase A (1), lysozyme (2)
and
α
-chymotrypsinogen A (3)
Sample load:
100µL
Gradient:
60 min linear gradient from buffer A to B
Buffer A:
1.8mol/L (NH
4
)
2
SO
4
+ 0.1mol/L
sodium phosphate (pH 7.0)
Buffer B:
0.1mol/L sodium phosphate (pH 7.0)
Linear velocity: 136cm/hr
Temperature:
25°C
Detector:
UV @ 280nm
0
10
20
30
40
50
60
70
UV @ 280 nm (mAU)
Elution volume (mL)
Toyopearl Phenyl-600M
Toyopearl PPG-600M
Toyopearl Butyl-600M
1
2
3
0
50
100
150
Column: 7.5 mm ID X 7.5 cm L; Sampl : 1 g/L RNase A (1),
lysozyme (2) and
a
-chymotrypsinogen A (3)
Sample load: 100 µL; Gradient: 60 min linear gradient from buffer A to B;
Buffer A: 1.8 mol/L (NH
4
)
2
SO
4
+ 0.1 mol/L sodium phosphate (pH 7.0); Buf-
fer B: 0.1 mol/L sodium phosphate (pH 7.0)
Linear velocity: 136 cm/h (1.0 mL/min); Temperature: 25 °C
Detector: UV @ 280 nm
figure 8
Pressure-flow curve for Toyopearl Phenyl-650 resins
of various particle size
Flow (cm/h)
0
Column:
Eluent:
0
Phenyl-650M
Pressure (bar)
Phenyl-650C
Phenyl-650S
Toyopearl Phenyl-650C, M and S, 25mm ID x 25cm
2mol/L (NH
4
)
2
SO
4
1.5
2
2.5
3
400
600
800
200
Column: TOYOPEARL Phenyl-650C, M and S, 25 mm ID x 25 cm L
Mobile phase: 2 mol/L (NH
4
)
2
SO
4
Pressure-flow curve for TOYOPEARL Phenyl-650 resins
of various particle size
figure 9
0
0.05
0.1
0.15
0
10
20
30
40
50
C/C
0
IgG loaded (g/L)
Breakthrough curves of polyclonal IgG on various HIC resins
Phenyl-600M
Phenyl Agarose
Butyl-600M
Phenyl-650M
PPG-600M
Column:
7.8mm ID X 20cm
Sample:
polyclonal human IgG
Binding buffer: 1g/L IgG in 0.8mol/L (NH
4
)
2
SO
4
+ 0.1mol/L
sodium phosphate (pH 7.0)
Linear velocity: 300cm/hr
Temperature:
25°C
Detector:
UV @ 280nm
DBC was calculated at 10% of breakthrough.
Column: 7.8 mm ID X 20 cm L; Sample: polyclonal human IgG
Binding buffer: 1 g/L IgG in 0.8 mol/L (NH
4
)
2
SO
4
+ 0.1 mol/L
sodium phosphate (pH 7.0); Linear velocity: 300 cm/h
Temperature: 25 °C; Detector: UV @ 280 n
DBC was calculated at 10% of breakthrough.
Breakthrough curves of polyclonal IgG on various
HIC Resins
Comparison of TOYOPEARL 600M series resins
