IEC
54
TOSOH BIOSCIENCE
APPLICATIONS - TSKgel CATION EXCHANGE COLUMNS
TSKgel SP-NPR
TSKgel SP-NPR columns provide fast separations due to their small
(2.5 µm) spherical particles. A purity check of adeno-associated virus,
commonly used in gene therapy research, on a TSKgel SP-NPR column
is shown in
Figure 11
.
This 10 minute HPLC method replaces an existing
assay that took two days.
Column: TSKgel SP-NPR, 4.6 mm ID x 3.5 cm L; Sample: purified adeno-
associated virus; Elution: A. 50 mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L
MgCl, pH 7.5; B. 50mmol/L HEPES, 1 mmol/L EDTA, 5 mmol/L MgCl, pH
7.5 with 0.5 mol/L NaCl; linear gradient from 20 % to 100 % B in 10 column
volumes; Flow rate: 1 mL/min; Detection: UV @ 280 nm
Columns: TSKgel BioAssist S, 4.6 mm ID x 5 cm L, PEEK
Competitor S 5mm ID x 5cm; Elution: 20 min (TSKgel) or 30 min (Competitor
S) linear gradient of NaCl from 0 to 0.5 mol/L in 20 mmol/L sodium phosphate
buffer, pH 7.0; Flow rate: 0.8 mL/min for TSKgel; 1.0 mL/min for Competitor S
Detection: UV @ 280 nm; Temperature: 25°C;
Sample: crude bromelain (C4882, Sigma), 1 mg in 100 μL
TSKgel Bioassist S
Especially designed for the separation of large biomolecules such
as antibodies, the very large pores of the TSKgel BioAssist columns
offer high capacity and resolution at a low column pressure drop. The
polymerization technique used to construct these columns results in a
homogenous distribution of ion exchange groups without significantly
reducing pore size. TSKgel BioAssist S is suitable for use in systems that
are designed for HPLC, laboratory, or semi-preparative applications. The
large pore size of the TSKgel BioAssist S resin provides high dynamic
capacity due to novel bonded phase design.
Figure 12
demonstrates
these features for the analysis of bromelain, a proteolytic enzyme that
is used as a nutritional supplement. Bromelain is a basic glycoprotein
with a MW of 33 kDa and a pI of 9.55.
IgM is known to possess unique and beneficial characteristics relative
to other immunoglobulin classes; it is a large molecule comprised of
five IgG subunits, resulting in a relatively unstable and difficult to purify
protein. Unlike single chain antibodies, IgMcannot be purified by Protein
A (an affinity material commonly used for its high binding capacity and
excellent selectivity for antibodies) due to steric hindrance. Alternative
affinity methods have been developed with thiophillic absorbents but
these methods often result in low binding capacity. An alternative
purification method of IgM by ion exchange chromatography using a
TSKgel BioAssist S column was developed. As shown in
Figure 13
baseline separation of IgM from other contaminants is achieved using a
0.3 mol/L NaCl step gradient after elution of albumin.
Column: TSKgel BioAssist S, 7 µm, 4.6 mm ID x 5 cm L;
Mobile phase: 20 mmol/L sodium phosphate buffer, pH 6.0;
Gradient: 0 mol/L - 0.3 mol/L NaCl (5 min), 0.3 mol/L - 0.5 mol/L NaCl (10 min);
Flow rate: 1 mL/min; Detection: UV@ 280 nm; Sample: 500 µL of 9.5 mg/mL IgM
in mouse ascites fluid; shaded peaks represent albumin and IgM respectively
figure 11
Analysis of purified AAV with Tskgel SP-NPR
Analysis of purified AAV with T Kgel SP-NPR
-5
0
5
10
15
20
2.5
5
7.5
10
0
Minutes
mAU
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel SP-NPR, 4.6mm ID x 3.5cm
purified adeno-associated virus
A. 50mmol/L HEPES, 1mmol/L EDTA, 5mmol/L MgCl, pH 7.5;
B. 50mmol/L HEPES, 1mmol/L EDTA, 5mmol/L MgCl, pH 7.5 with
0.5mol/L NaCl; linear gradient from 20% to 100% B in
10 column volum s
1mL/min
UV @ 280nm
figure 12
Bromelain Analysis on TSKgel Bioassist S and competitor S Columns
0
100
200
300
400
500
0
5
10
15
20
25
Competitor S
BioAssist S
Bromelain Analysis on TSKgel Bioassist S and Competitor S Columns
Columns:
TSKgel BioAssist S, 4.6mm ID x 5cm, PEEK
Compet tor S 5mm ID x 5cm
Elution:
20 min (TSKgel) or 30 min (Competitor S) linear gradient of NaCl
from 0 to 0.5mol/L in 20mmol/L sodium phosphate buffer, pH 7.0
Flow Rate:
0.8mL/min for TSKgel; 1.0mL/min for Competitor S
Detection:
UV @ 28 nm
Temperature: 25°C
Sample:
crude bromelain (C4882, Sigma), 1mg in 100µL
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1
2
0
0.1
0.2
0.5
0.4
0.3
0.6
0.7
0.8
Abs (280mm)
NaCl (M) in eluent
Retention time (minutes)
0
5
10
15
TSKgel
Mobile
Gradien
Flow Ra
Detecti
Sample:
figure 13
Analysis of IgM
