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SEC
37
Applications of TSKgel SW-type Gel Filtration columns
Comparing TSKgel SW, SW
XL
and SuperSW Gel Filtration
Columns
Figure 1
&
Figure 2
show the increased resolution and sensitivity of
the TSKgel SuperSW columns compared to TSKgel SW
XL
columns. This
is due to the smaller particle size (4 vs. 5 µm) coupled with a narrow
column (4.6 mm ID).
Analysis of protein aggregation
TSKgel G3000SW
XL
columns are the industry standard for agregation
analysis in quality control of monoclonal antibodies (mAbs).
Figure 3
shows the analysis of mAb Aggregates with UV, refractive index (RI) and
multi angle light scattering (MALS) detection. When the protein analysis
needs to be performed in a metal free environment, the BioAssistSW
series offers TSKgel SW packings in PEEK housings, featuring the same
performance as stainless steel columns.
figure 3
SEC-Mals-UV-RI analysis of MAB aggregates
Comparison of TSKgel SuperSW3000 and TSKgel G3000SW
XL
for the separation of proteins
Minutes
Column:
Sample:
Elution:
Flow Rate:
Temp:
Detection:
A.TSKgel G3000SW
XL
, 7.8mm ID x 30cm;
B. TSKgel SuperSW3000, 4.6mm ID x 30cm
5µL of a mixture of 1. thyroglobulin, 0.5mg/mL (660,000Da);
5. p-aminobenzoic acid, 0.01mg/mL (137Da)
0.1mol/L NaSO
4
in 0.1mol/L in phosphate buffer with 0.05%
NaN
3
, pH 6.7
1.0mL/min for G3000SW
XL
; 0.35mL/min for SuperSW3000
25
°
C
UV @ 220nm
0
10
20
30
40
50
3 5 7 9 11 13 15
0
10
20
30
40
50
5
Rs: 10.7
4
Rs: 4.0
2
3
Rs: 3.5
1
5
4
3 2
1
RS: 8.4
RS: 3.0
RS: 3.3
A. G3000SW
XL
B. SuperSW3000
mV
4. ribonuclease A, 1.5mg/mL (12,600Da);
Minutes
3 5 7 9 11 13 15
2.
γ
-globulin, 1.0mg/mL; (150,000 Da);
3. ovalbumin, 1.0mg/mL (43,000Da);
Column: A. TSKgel G3000SW
XL
, 7.8 mm ID x 30 cm L;
B. TSKgel SuperSW3000, 4.6 mm ID x 30 cm L;
Sample: 5
μ
L of a mixture of 1. thyroglobulin, 0.5 mg/mL (660,000 Da);
2.
g
-globulin, 1.0 mg/mL (150,000 Da); 3. ovalbumin, 1.0 mg/mL (43,000 Da);
4. ribonuclease A, 1.5 mg/ L (12,60 Da); 5.
r
-aminobenzoic acid,
0.01 mg/mL (137 Da);
Elution: 0.1 mol/L NaSO
4
in 0.1 mol/L in phosphate buffer with 0.05 % NaN
3
,
pH 6.7; Flow Rate: 1.0 mL/min for G3000SW
XL
; 0.35 mL/min for SuperSW3000;
Temp: 25°C; Detection: UV @ 220nm
3
5
7
9 11 13 15
0
10
20
30
40
50
60
Minutes
mV
Column:
Sample:
Inj. Volume:
Elution:
Flow Rate:
Temp:
Detection:
A. TSKgel G2000SW
XL
, 7.8mm ID x 30cm;
B. TSKgel SuperSW2000, 4.6mm ID x 30cm
1. thyroglobulin (0.2mg/mL); 2. albumin (1.0mg/mL);
3. ribonuclease A (1.0mg/mL); 4. p-aminobenzoic acid (0.01mg/mL)
5µL
0.1mol/L phosphate buffer + 0.1mol/L Na
2
SO
4
+ 0.05% NaN
3
(pH 6.7)
0.35mL/min for SuperSW2000; 1.0mL/min for G2000SW
XL
25
°
C
UV @ 280nm
1
Rs:4.7 Rs:4.6
Rs:11.9
2
3
4
G2000SW
XL
3
5
7
9 11 13 15
0
10
20
30
40
50
60
Minutes
mV
1
Rs:4.8
Rs:6.2
Rs:15.6
2
3
4
SuperSW2000
Comparison of TSKgel SuperSW2000 and TSKgel G2000SW
XL
for the separation of proteins
A.
B.
Column: A. TSKgel G2000SW
XL
, 7.8 m ID x 30 cm L;
B. TSKgel SuperSW2000, 4.6 mm ID x 30cm L;
Sample: 1. thyroglobulin (0.2 mg/mL); 2. albumin (1.0 mg/mL); 3. ribonucle-
ase A (1.0 mg/mL); 4. p-aminobenzoic acid (0.01 mg/mL); Inj. Volume: 5 μL;
Elution: 0.1 mol/L phosphate buffer + 0.1 mol/L Na
2
SO
4
+ 0.05 % NaN
3
(pH 6.7);
Flow Rate: 0.35 mL/min for SuperSW2000; 1.0 mL/min for G2000SW
XL
;
Temp: 25°C; Detection: UV @ 280nm
0.15
0.10
0.05
0.00
0.0
5.0
10.0
time (min)
detector voltage (V)
Multimer
Dimer
Monoclonal
Column: TSKgel G3000SW
XL
column, 5 µm, 7.8 mm ID x 30 cm L
Sample: monoclonal antibody, Inj.volume: 20 µL;
Mobile phase: phosphate buffered saline (PBS); Flow rate: 1 mL/min;
Detection: MALS (red), refractive index (blue) & UV @ 280 nm (green);
HPLC System: LC-20A prominence, Shimadzu;
MALS detector: miniDAWN
TM
TREOS, Wyatt Techn. Corp.
figure 1
Comparison of tskgel super sw3000 and tskgel g3000sw
XL
for the
separation of proteins
figure 2
Comparison of tskgel super sw2000 and tskgel g3000sw for the
separation of Proteins