SEC
14
figure 4
Comparison of resolution of mAb monomer and dimer
4
5
6
7
8
9
10
Retention time (minutes)
Detector response (mAU)
Dimer
Monomer
A. TSKgelSuperSW
mAbHR
B. BrandA
(3µm)
C. BrandB
(5µm)
D. BrandC
(5µm)
E. BrandD
(5µm)
Rs = 2.01
Rs = 1.95
Rs = 1.56
Rs = 1.59
Rs = 1.33
Columns:
A. TSKgel SuperSW mAb HR, 4 µm, 7.8 mm ID
×
30 cm
B. Brand A, 3 µm, 7.8 mm ID
×
30 cm
C. Brand B, 5 µm, 7.8 mm ID
×
30 cm
D. Brand C, 5 µm, 8.0 mm ID
×
30 cm
E. Brand D, 5 µm, 8.0 mm ID
×
30 cm
Mobile phase: 200 mmol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate:
1.0 mL/min
Detection:
UV @ 280 nm
Temperature:
25 ˚C
Injection vol.:
10 µL
Samples:
IgG (human polyclonal) (1.0 g/L)
Columns: A. TSKgel SuperSW mAb HR, 4 µm, 7.8 mm ID × 30 cm;
B. Brand A, 3 μm, 7.8 mm ID × 30 cm; C. Brand B, 5 μm, 7.8 m ID × 30 cm;
D. Brand C, 5 μm, 8.0 mm ID × 30 cm; E. Brand D, 5 μm, 8.0 mm ID × 30 cm
Mobile phase: 200 mmol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate: 1.0 mL/min; Detection: UV @ 280 nm
Temperature: 25 ˚C; Injection vol.: 10 μL
Sample: IgG (human polyclonal), 1.0 g/L
0
20
40
60
80
100
120
0
1
2
3
4
5
6
7
8
Retention time (minutes)
Detector response (UV)
TSKgel SuperSW mAbHTP
(4.6mmID x15cm)
monoclonal antibody-2
monomer
dimer
trimer
aggregates
Flow rate: 0.50 mL/min
Pressure: 5.0 MPa
Rs(dimmer/monomer)=1.91
Flow rate: 0.35 mL/min
Pressure: 3.6 MPa
Rs(dimmer/monomer)=2.13
Column:
TSKgel SuperSW mAb HTP (4.6 mm ID
×
15 cm)
Mobile phase: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN
3
Flow rate:
0.50 mL/min, 0.35 mL/min
Detection:
UV @ 280 nm
Temperature:
25 ˚C
Sample:
monoclonal antibody-2 (mouse-human chimeric
IgG, Erbitux), 5 µL
Column: TSKgel SuperSW mAb HTP, 4 µm, 4.6 mm ID × 15 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate: 0.50 mL/min, 0.35 mL/min; Detection: UV @ 280 nm
Temperature: 25 ˚C; Sa ple: monoclonal antibody-2
(mouse-human chimeric IgG, Erbitux
®
), 5 µL
figure 3
High speed separation of therapeutic mAb
Applications of TSKgel SW-type Gel Filtration columns
ANALYSIS OF MONOCLONAL ANTIBODIES:
The TSKgel SuperSW mAb size exclusion series consists of three
specialized columns designed for the separation and analysis of
monoclonal antibodies (mAb). Compared to competitive columns, these
new stainless steel, silica-based TSKgel columns offer reduced lot-to-
lot variation, long column life, reduction of unspecified adsorption, and
superior recovery of aggregates. TSKgel mAb columns are compatible
with both HPLC and UHPLC systems.
These columns are available within the TSKgel SW mAb column line:
• TSKgel SuperSW mAb HR
• TSKgel SuperSW mAb HTP
• TSKgel UltraSW Aggregate
TSKgel SuperSW mAb HR and SuperSW mAb HTP both contain 4 μm
particles. The HR designation represents the high resolution analysis
of mAb monomer, dimer, and fragments, while the HTP stands for “high
throughput” due to the smaller dimensions (4.6 mm ID × 15 cm). The
TSKgel UltraSW Aggregate column is packed with particles featuring
a smaller particle size, 3 μm, and slightly larger pore size. It offers high
resolution separation of mAb multimers.
These columns utilize a unique technology, which produces a shallow
calibration curve in the molar mass region of a typical antibody. The
calibration curve for the TSKgel SuperSW mAb HR column is similar
to that of TSKgel G3000SW
XL
. It has a shallower slope than the TSKgel
UltraSW Aggregate column around the molar mass range of
g
-globulin
resulting in a higher resolution for that mass range.
HIGH SPEED ANALYSIS OF THERAPEUTIC mAb
A shorter column length allows the TSKgel SuperSW mAb HTP column
to provide fast and efficient run times in the high resolution separation
of a mAb monomer and dimer.
FIGURE 3
shows no loss in resolution
in the analysis of a therapeutic mAb at a 0.50 mL/min flow rate and an
increased pressure of 5.0 MPa.
HIGH RESOLUTION SEPARATION OF MONOMER & DIMER
FIGURE 4
demonstrates the superior resolution of the TSKgel SuperSW
mAb HR column compared to four competitive columns in the analysis of
a mAb monomer and dimer. TSKgel SuperSW mAb HR shows excellent
resolution of gamma-globulin dimer and monomer.
DURABILITY OF SuperSW mAb COLUMNS
FIGURE 5
demonstrates the good durability of the TSKgel SuperSW
mAb HR column through the reproducibility of resolution for a
γ
-globulin
sample injection.
figure 5
High durability of TSKgel SuperSW mAb HR column
Column:
TSKgel SuperSW mAb HR, 4 µm, 7.8 mm ID × 30 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate:
0.8 mL/min
Detection:
UV @ 280 nm
Injection vol.:
10 µL
Samples:
1.
g
-Globulin
2. Cytochrome C
3. DNP-L-Alanine
4
Inj. 599
1
3
2
Rs= 1.74
Rs= 1.79
Rs= 1.87
Inj. 303
Inj. 3
0
50
100
6
8
10
Detector response (mV)
Retention time (minutes)
TSKgel SuperSW mAb HR exhibits good durability
of Rs for gamma-globulin sampl injection
12
14
Column: TSKgel SuperSW mAb HR, 4 µm, 7.8 mm ID × 30 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate: 0.8 mL/min; Detection: UV @ 280 nm
Injection vol.: 10 μL
Samples: 1.
g
-Globulin; 2. Cytochrome C; 3. DNP-L-Alanine
