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SEC
38
Applications of TSKgel SW-type Gel Filtration columns
A
nalysis of antibody-fusion proteins
During method development, many variables are examined to ensure
method robustness. Factors such as elution profile, peak shape, and
recovery are required to be consistent. During a method re-qualification
several variables were investigated to eliminate non-specific binding
and increase the robustness of an established QC method using a
TSKgel SuperSW3000 column. As shown in
Figure 4
, excessive peak
tailing of “fusion protein 1” is evident with the use of 0.2 mol/L NaCl
(chromatogram c). Additionally, the expected protein dimer and trimer
aggregates are not visible. By switching from 0.2 mol/L sodium chloride
to 0.2 mol/L of the more chaotropic sodium perchlorate salt, together
with a two-fold reduction in the buffer concentration, less peak tailing
and distinct peaks for the dimer and trimer species of mAb 1 resulted
(chromatogram B). Doubling the perchlorate concentration to 0.4 mol/L
provided further improvement in the peak shape of fusion protein 1
and associated aggregate species (chromatogram A).
Figure 4B
is an
enlargement of the baseline region, showing an improved peak shape
of the dimer and trimer aggregates with the use of 0.4 mol/L NaClO
4
.
Column: TSKgel SuperSW3000, 4 μm, 4.6 mm ID x 30 cm L;
Mobile phase: c: 0.4 mol/L NaClO
4
, 0.05 mol/L NaH
2
PO
4
, b: 0.2 mol/L NaClO
4
,
0.05 mol/L NaH
2
PO
4
, a: 0.2 mol/L NaCl, 0.1 mol/L NaH
2
PO
4
; Flow rate: 0.35 mL/
min; Detection: UV@214nm; Injection vol.: 5 μL; Samples: antibody fusion
protein
Membrane Proteins
The effect of different concentrations of surfactant on the separation
of membrane proteins is seen in
Figure 5
. As the concentration of
octaethyleneglycol dodecylether increases to 0.05%, the main peak
becomes sharper and recovery increases.
Enzymes
Mobile phase conditions in GFC are optimized to ensure little or
no interaction of the sample with the packing material. This gentle
technique allows for high recovery of enzymatic activity. A crude
sample of glutathione S-transferase was separated in only 15 minutes
on a TSKgel G3000SW
XL
column and activity recovery was 98% and
89%, respectively. The elution profile of the separation in
FIGURE 6
shows that all of the activity eluted in a norrow band of about 1.5 mL.
Column: TSKgel G3000SW, 7.5mm ID x 60 cm L; Sample: Membrane protein
from a crude extract from rat liver microsome; Elution: (0.2 mol/L sodium
chloride + 20 % glycerol + octaethyleneglycol dodecylether) in 50 mmol/L
phosphate buffer, pH 7.0.
Note: concentration of surfactant: (1) 0.005 %, (2) 0.01 %, (3) 0.025 %, (4) 0.05 %;
Flow Rate: 1.0 mL/min; Detection: UV @ 280 nm
Column: TSKgel G3000SW
XL
5 μm, (7.8 mm ID x 30 cm L); Sample: crude
glutathione S-transferase from guinea pig liver extract, 0.7 mg in 0.1 mL;
Elution: 0.3 mol/L NaCl in 0.05 mol/L phosphate buffer, pH 7;
Flow Rate: 1.0mL/min; Detection: UV@220 nm (solid line) and enzyme assay
tests (dashed line); Recovery: enzymatic activity recovered was 89 %
Separation of crude protein samples on TSKgel G3000SW
XL
A. crude peroxidase
B. crude glutathione S-transferase
Minutes
Minutes
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel G3000SW
XL
, 5µm, 7.8mm ID x 30cm
A. crude peroxidase from Japanese radish,
0.15mg in 0.1mL
B. crude glutathione S-tr nsferase from guinea
p g liv r extract, 0.7mg i 0.1mL
0.3m l/L NaCl in 0.05mol/L phosphat buffer, pH 7
1.0mL/min
UV @ 220nm (solid line) and enzyme assay
tests (dashed line)
0
10
15
0
10
15
5
5
figure 6
Separation of crude protein sample on tskgel g3000sw
XL
figure 5
Separation of membrane protein by SEC with different surfactant
concentration in the eluent
figure 4
Overlays of Antibody fusion protein analysis
mAU
1000
800
600
400
200
0
2
4
6
8
10
12
A
A
B
B
C
C
mAU
70
60
50
40
30
20
10
0
-10
4
5
6
7
8
9
A.
B.
fusion
protein1
Sep rati n of embrane protein by SEC with different
surfactant concentration in the eluent
Minutes
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel G3000SW, 7.5 m ID x 60cm
Membrane protein fro a crude extract from
rat liver microsome
(0.2mol/L sodium chloride + 20% glycerol +
ctaethyleneglycol dodecylether) in 50mmol/L
phosphate buffer, pH7.0. Note: concentration
of surfactant: (1) 0.005%, (2) 0.01%, (3) 0.025%,
(4) 0.05%
1.0mL/min
UV @ 280nm
10
20
30
40
(1)
(2)
(3)
(4)