12
IEC
IEC
TSKgel Bioassist SERIES
APPLICATIONS WITH TSKgel BioAsisst ANION
EXCHANGE COLUMNS
Comparison of Dynamic Binding Capacity
Table 2 shows typical dynamic binding capacities on Bio-
Assist Q relative to competitive products.
Applications with TSKgel BioAssist Cation
Exchange Columns
Bromelain Anaylsis on TSKgel BioAssist S and Competitor
S Column
Figure 12 shows 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.
Analysis of IgM
IgM is known to possess unique and beneficial charac-
teristics 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, IgM cannot be purified by Protein A
(affinitymaterialcommonlyusedforitshighbindingcapacity
and excellent selectivity for antibodies) due to steric hin-
drance. 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. Figure 13 shows the
baseline separation of IgM from other contaminants using
a 0.3 mol/L NaCl step gradient after elution of albumin.
Binding capacity (mg/mL)
BioAssist Q SuperQ Conv.
Conv.
-5PW Q type
Q type
Protein
prod. A prod. B
Thyroglobulin
77.4
22.9
20.2
1.8
Monoclonal IgG
1
57.8
43.3
46.7
47.7
Human Serum
83.1
78.9
48.2
48.8
Albumin
Trypsin Inhibitor
84.3
92.8
51.8
57.8
Columns:
TSKgel BioAssist Q and TSKgel SuperQ-5PW (4.6 mm ID x 1 cm);
Conventional Q type product A and B (4.6 mm ID x 1 cm)
Solvent:
20 mmol/L Tris-HCl buffer, pH 8.0;
Flow rate:
0.38 mL/min; Det.: UV @ 280 nm
*Capacity was determined at 10% height of the breakthrough curve; UV 280 nm.
TABLE II
comparison of dynamic binding capacities
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
Retention time
bromelain analysis on TSKgel BioAssist s & competitor s
columns
Columns: TSKgel BioAssist S, 4.6 mm ID x 5 cm L, PEEK
Competitor S 5 mm ID x 5 cm L; 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
figure 12
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 p
Gradient
Flow Rat
Detectio
Sample:
analysis of IgM
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 13
