TOSOH BIOSCIENCE
TSKgel SW columns for mAb separation
TSKgel SuperSW mAb HR/HTP
TSKgel UltraSW Aggregate
TOSOH BIOSCIENCE
Structure of Antibodies
2
Antibodies are immune system-related proteins called immunoglobulins (IgG).
Each IgG consists of four polypeptides– two heavy chains and two light chains
joined to form a "Y" shaped molecule.
Monoclonal antibodies (mAb) are monospecific antibodies produced by
clones of a unique parent cell.
TOSOH BIOSCIENCE 3 3
• Therapeutic antibodies and other recombinant
proteins are widely used for therapeutic treatment.
• Heterogeneity evaluation is essential during
development, stability testing and quality control of
the final product.
• Analysis of aggregates and denatured proteins is
important because they might increase
immunogenicity of the product.
Therapeutic Proteins
TOSOH BIOSCIENCE
Analysis of mAb Heterogeneity
Aggregation, dimer, fragmentation >>> SEC
>>> HIC
Charge-related heterogeneity
Deamidation, termini modification etc. >>> IEC
Oxidation >>> HIC/RPC
Glycosylation
Full length mAb >>> IEC
Glycan composition >>> HILIC
Glycation >>> AFC/IEC
4
TOSOH BIOSCIENCE
SEC Separation for Antibodies - Demands
• Higher resolution of mAb monomer and dimer/fragment
• High-throughput separation of mAb monomer and dimer
• Higher resolution of mAb aggregates
• Reduction of lot-to-lot variation
• Increase of column life time
• Reduction of unspecific adsorption
• Good recovery, especially for aggregates
5
TOSOH BIOSCIENCE 6
New SW columns for mAb separation
High resolution analysis of mAb monomers, dimers and
fragments:
SuperSW mAb HR (4µm; 7.8 mm ID x 30 cm)
High throughput analysis of mAbs:
(particularly for separation of dimer and monomer)
TSKgel SuperSW mAb HTP (4µm; 4.6 mm ID x 15 cm)
High resolution analysis of mAb aggregates, multimers:
TSKgel UltraSW Aggregate (3µm; 7.8 mm ID x 30 cm)
TOSOH BIOSCIENCE 7
Characteristics of New Stationary Phases
ColumnTSKgel SuperSW
mAb HR
TSKgel SuperSW
mAb HTP
TSKgel UltraSW
Aggregate
Column dimension 7.8 mm ID x 30 cm 4.6 mm ID x 15 cm 7.8 mm ID x 30 cm
Base material Silica gel
Functional group Diol
Particle size 3 μm
Pore size 30 nm
Separation range(for globular proteins)
10,000 - 2,000,000 Da
ApplicationsSeparation of mAb
monomer and dimer
Fast separation of mAb
monomer and dimer
(UHPLC compatible).
Separation of mAb
aggregates.
10,000 - 500,000 Da
Silica gel
Diol
4 μm
25 nm
TOSOH BIOSCIENCE
Product line and specification
P/N Product Column size
(mmI.D. x cm)
Theoretical
plates
Asymmetry
factor
0022854 TSKgel SuperSW mAb HR 7.8 x 30 >= 30,000 1.2 – 1.8
0022855 TSKgel SuperSW mAb HTP 4.6 x 15 >= 15,000 0.8 – 1.4
0022856 TSKgel UltraSW Aggregate 7.8 x 30 >= 35,000 1.2 – 1.8
8
Analytical columns
P/N Product Column size
(mmI.D. x cm)
Coresponding
analytical
column (P/N)
0022857 TSKgel guardcolumn SuperSW mAb 6.0 x 4 0022854
0022858 TSKgel guardcolumn SuperSW mAb 3.0 x 2 0022855
0022859 TSKgel guardcolumn UltraSW 6.0 x 4 0022856
Guardcolumns
TOSOH BIOSCIENCE
Chromatograms – Protein Standard
9
Columns: TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm),
TSKgel SuperSW mAb HR(7.8 mm ID x 30 cm),
TSKgel G3000SWXL (7.8 mm ID x 30 cm)
Mobile phase : 200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 1.0 mL/min
Temperature: 25℃
Detection: UV @ 280 nm
Injection vol.: 10 μL
Samples: 1 thyroglobulin (MW 640,000) (0.5 g/L)
(1’ thyroglobulin multimers)
2 γ-globulin (MW 155,000) (1.0 g/L)
(2’ γ-globulin dimer)
3 ovalbumin (MW 47,000) (1.0 g/L)
4 ribonuclease A (MW 13,700) (1.5 g/L)
5 p-aminobenzoic acid (MW 137) (0.01 g/L)
0
50
100
150
200
250
0 2 4 6 8 10 12 14
Time [min]
UV
280 n
m
TSKgel
G3000SWXL
(5μm, 25nm)
TSKgel
SuperSW mAb HR
(4μm, 25nm)
TSKgel
UltraSW Aggregate
(3μm, 30nm)
1 2
3
4 5
2'1'
1 23
4 5
2'1'
• SuperSW mAb HR shows higher resolution
between gamma-globulin dimer and monomer,
gamma-globulin monomer and ovalbumin. It
has a calibration curve similar to G3000SWXL
• UltraSW Aggregate shows higher resolution
between thyroglobulin monomer and dimer. It
has a wider separation window of thyroglobulin
multimer region
Vo
Vo
TOSOH BIOSCIENCE
Calibration Curves - Protein Standard
TSKgel UltraSW Aggregate:
Covers separation range up to around 2
million Da. This implies better resolution
of aggregate/multimer of mAb.
TSKgel SuperSW mAb HR:
Shows a calibration curve similar to
TSKgel G3000SWXL and a shallower
slope than TSKgel UltraSW around Mw
range of gamma-globulin
10
1
2
3
4
5
6
7
4 6 8 10 12 14
Elution time [min]
Log M
W
UltraSW Aggregate
SuperSW mAb HR
G3000SWXL
8.6 x 105 Da (SuperSW mAb HR)
2.5 x 106 Da (UltraSW Aggregate)
Exclusion limit (globular protein):
← γ-Globulin (MW 155,000)
← Thyroglobulin (MW 640,000)
← Ovalbulin (MW 47,000)
← Thyroglobulin dimer
← Thyroglobulin trimer
← Ribonuclease A
(MW 13,700)
p-Aminobenzoic acid
(MW 137) →
TOSOH BIOSCIENCE 11
Chromatograms of Standard Proteins
Column dim.: A-E: 7.8 mm ID x 30 cm
F&G: 8.0 mm ID x 30 cm
H: 4.6 mm ID x 15 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN3
Flow rate: A-G: 1.0 mL/min H: 0.35 mL/min
Temperature: 25℃
Detection: UV @ 280 nm
Injection vol.: A-G: 10 μL H: 3.5 μL
Samples: 1 thyroglobulin (MW 640,000) (A-G: 0.5 g/L H: 2.0 g/L)
(1’) thyroglobulin oligomer
2 γ-globulin (MW 155,000) (A-G: 1.0 g/L H: 1.5 g/L)
3 ovalbumin (MW 47,000) (A-G: 1.0 g/L H: 1.5 g/L)
4 ribonuclease A (MW 13,700) (1.5 g/L)
5 p-aminobenzoic acid (MW 137) (0.01 g/L)
0 1 2 3 4 5 6 7
Time [min]
AB
S @
280nm 1 2 3 4
5
(1')
H. SuperSW
mAb HTP
New TSKgel mAb SEC columns show superior
performance compared to competitor columns.
2 4 6 8 10 12 14
Time [min]
AB
S @
280 n
m [
AU
]
12 3
4 5
(1')A. SuperSW
mAb HR
B. UltraSW
Aggregate
C. G3000SWXL
D. Brand A
(3 μm)
E. Brand B
(5 μm)
F. Brand C
(5 μm)
G. Brand D
(5 μm)
TOSOH BIOSCIENCE
Resolution of mAb Dimer and Monomer
12
Column: A. TSKgel SuperSW mAb HR (7.8 mm ID x 30 cm)
B. TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm)
C. TSKgel G3000SWXL (7.8 mm ID x 30 cm)
D. Brand A (3μm) (7.8 mm ID x 30 cm)
E. Brand B (5μm) (7.8 mm ID x 30 cm)
F. Brand C (5μm) (8.0 mm ID x 30 cm)
G. Brand D(5μm) (8.0 mm ID x 30 cm)
H. TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
Mobile phase: 200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: A-G. 1.0 mL/min, H. 0.35 mL/min
Temperature: 25℃;
Detection: UV @ 280 nm
Injection vol.: A-G. 10 μL H. 3.5 μL
Samples: IgG (human polyclonal) (A-G: 1.0 g/L, H: 4.5 g/L)
2.0 2.5 3.0 3.5 4.0 4.5
Time [min]
UV
280 n
m
H. SuperSW
mAb HTP
Rs = 1.69
TSKgel SuperSW mAb HR:
Superior resolution of gamma-globulin dimer and monomer
TSKgel SuperSW mAb HTP:
Good resolution of gamma-globulin dimer and monomer in
half of the analysis time of conventional columns
YMC-Pack Diol-300
4 5 6 7 8 9 10
Time [min]
UV
280 n
m
dimer
monomer
A. SuperSW
mAb HR
B. UltraSW
Aggregate
C. G3000SWXL
D. Brand A
(3 μm)
E. Brand B
(5 μm)
F. Brand C
(5 μm)
G. Brand D
(5 μm)
Rs = 2.01
Rs = 1.66
Rs = 1.64
Rs = 1.95
Rs = 1.56
Rs = 1.59
Rs = 1.33
TOSOH BIOSCIENCE
Separation of Therapeutic Antibody
13
Rs(3mer/2mer) =1.40
Rs(2mer/monomer) =2.89
Rs(3mer/2mer) =1.32
Rs(2mer/monomer) =3.11
Df (mono./fragment) =0.095
Rs(3mer/2mer) =0.96
Rs(2mer/monomer) =2.32
TSKgel SuperSW mAb HR:
superior resolution of mAb dimer,
monomer and fragment
TSKgel UltraSW Aggregate:
Superior resolution of mAb
trimer and dimer
fragment
Column: TSKgel UltraSW Aggregate
(7.8 mm ID x 30 cm)
TSKgel SuperSW mAb HR
(7.8 mm ID x 30 cm)
Eluent : 0.2 mol/L phosphate (pH 6.7) + 0.05% NaN3
Flow rate: 0.8 mL/min
Detection: UV @ 280 nm
Temp.: 25℃
Sample: monoclonal antibody-2
(mouse-human chimeric IgG, Erbitux), 10 uL
TOSOH BIOSCIENCE 14
Column: TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
Elution: 0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 0.50 mL/min, 0.35 mL/min;
Detection: UV @ 280 n;m Temp.: 25℃;
Sample: 5 uL monoclonal antibody-2 (mouse-human chimeric IgG, Erbitux)
High Speed Separation of Therap. Antibody
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8
Time [min]
UV
28
0 n
mTSKgel
SuperSW mAb HTP
(4.6mmID x 15cm)
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
TSKgel SuperSW mAb HTP
showed superior resolution of mAb
dimer and monomer even at high flow
rate with low pressure drops
TOSOH BIOSCIENCE
Separation of IgG Aggregates
15
Columns: TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm),
TSKgel SuperSW mAb HR(7.8 mm ID x 30 cm),
TSKgel G3000SWXL (7.8 mm ID x 30 cm)
Mobile phase : 200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 1.0 mL/min
Temperature: 25℃
Detection: UV @ 280 nm
Injection vol.: 20 μL
Sample: IgG (human monoclonal, BI-MAb-02, 2.5 g/L)
(1 monomer, 2 dimer, 3 trimer, 4 tetramer, 5 multimers)
0
50
100
150
200
250
3 4 5 6 7 8 9 10
Time [min]
UV
280 n
m
TSKgel
G3000SWXL
(5μm, 25nm)
TSKgel
SuperSW mAb HR
(4μm, 25nm)
TSKgel
UltraSW Aggregate
(3μm, 30nm)
1
2
3
45
Vo Vo
TSKgel UltraSW Aggregate:
Wider separation window in
aggregate region (> trimer)
TOSOH BIOSCIENCE
Effect of Flow Rate on Multimer Separation
Pentamer was separated at lower flow rate,
< 0.4 mL/min on UltraSW Aggregate
16
0
10
20
30
40
50
4 6 8 10
Time [min]
UV
280 n
m
1.0 mL/min
tetramer
0
10
20
30
40
50
10 15 20 25
Time [min]
UV
280 n
m
0.4 mL/min
0
10
20
30
40
50
20 30 40 50
Time [min]
UV
280 n
m
0.2 mL/min
pentamer
Column: TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm)
Mobile phase : 200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 0.2~1.0 mL/min
Temperature: 25℃
Detection: UV @ 280 nm
Injection vol.: 10 μL
Sample: IgG (human monoclonal, MAb-02, 5 g/L)
TOSOH BIOSCIENCE
Effect of Flow Rate on Separation
The lower the flow rate, the higher the column performance
17
0
1
2
3
4
5
6
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Flow rate [mL/min]
Rs
dimer / monomer
trimer / dimer
tetramer / trimer
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Flow rate [mL/min]
TP
monomer
dimer
trimertetramer
Theoretical plates and flow rate Resolution and flow rate
TOSOH BIOSCIENCE
Effect of Flow Rate - TSKgel SuperSW mAb HR
18
Column: TSKgel SuperSW mAb HR (7.8 mm ID x 30 cm)
Mobile phase : 100 mmol/L phosphate buffer (pH 6.7)
+ 100 mmol/L Na2SO4 + 0.05% NaN3
Flow rate: 0.20~1.00 mL/min, Temperature: 25℃
Detection: UV @ 280 nm, Injection vol.: 10 μL
Samples: 1. Thyroglobulin (MW 640,000) (1.5 g/L)
2. γ-Globulin (MW 155,000) (1.5 g/L)
3. Ovalbumin (MW 47,000) (1.5 g/L)
4. Ribonuclease A (MW 13,700) (1.5 g/L)
5. p-Aminobenzoic acid (MW 137) (0.01 g/L)
van Deemter plot
0
5
10
15
20
25
30
35
40
45
50
55
60
0.0 0.5 1.0 1.5 2.0 2.5
u (Linear velocity) [cm/min]
HE
TP
[um
]
-200
-150
-100
-50
0
50
100
150
200
250
Thyroglobulin
γ-Globulin
Ovalbumin
Ribonuclease A
p-Aminobenzoic acid
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
2.5
0.0 0.5 1.0 1.5 2.0 2.5
u (Linear velocity) [cm/min]
Rs (
IgG
dim
er/
monom
er)
Resolution (IgG dimer and monomer)
Sample: IgG (human polyclonal) (4.5 g/L)
Other conditions were the same as
described left.
1.0 mL/min
0.5 mL/min
TOSOH BIOSCIENCE
Effect of Flow Rate - TSKgel SuperSW mAb HTP
19
Column: TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
Mobile phase : 200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
Flow rate: 0.143~0.476 mL/min; Temperature: 25℃
Detection: UV @ 280 nm, Injection vol.: 5 μL
Samples: IgG (human polyclonal) (4.5 g/L)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
0.0 0.1 0.2 0.3 0.4 0.5
Flow rate [mL/min]
Rs (
IgG
dim
er/
monom
er)
Rs =1.51
(0.476 mL/min)
Resolution (IgG dimer and monomer)
0
1
2
3
4
5
6
7
0.0 0.1 0.2 0.3 0.4 0.5
Flow rate [mL/min]
Pre
ssure
Dro
p [
MP
a]
5.6 MPa
Pressure drops
Standard flow rate Standard flow rate
High speed separation
Complete separation of IgG
dimer and monomer even with
high flow rate
Low pressure drops
Compatible to both UHPLC and
conventional HPLC
TOSOH BIOSCIENCE
Lot to Lot Reproducibility
0
50
100
150
200
250
4 6 8 10 12 14 16 18
Elution time (min)
mV
1
Lot. 501T
2 3
45
Lot. 502T
Lot. 503T
20
Column: TSKgel SuperSW mAb HR
(7.8 mm ID x 30 cm)
Eluent: 0.2 mol/L Phosphate Buffer (pH 6.7)
+0.05% NaN3
Flow rate: 0.8 mL/min
Detection: UV @ 280nm
Inj. volume: 10 μL
Sample: 1 Thyrobulobulin, 2 γ-Globulin, 3 Ovalbumin,
4 Ribonuclease A, 5 p-Aminobenzoic acid
TSKgel SuperSW mAb HR
exhibited good gel lot to lot
reproducibility
TOSOH BIOSCIENCE
Durability of TSKgel SuperSW mAb HR (1)
50
60
70
80
90
100
110
0 200 400 600
Inj. #
RT
(%)
50
60
70
80
90
100
110
0 200 400 600
Inj. #T
P (
%)
21
Column: TSKgel SuperSW mAb HR (7.8 mm ID x 30 cm)
Eluent: 0.2 mol/L Phosphate Buffer (pH 6.7)+0.05% NaN3
Flow rate: 0.8 mL/min Detection: UV280nm Inj. volume: 10 μL
Sample: 1 Thyrobulobulin, 2 γ-Globulin, 3 Ovalbumin, 4 Ribonuclease A, 5 p-Aminobenzoic acid
Change of retention time and theoretical plates for p-ABA
TOSOH BIOSCIENCE
Durability of TSKgel SuperSW mAb HR (2)
0
50
100
150
4 6 8 10 12 14
Elution time (min)
mV
1
Inj . 1
Inj . 301
Inj . 561
2 3
4
5
22
Column: TSKgel SuperSW mAb HR
(7.8 mm ID x 30 cm)
Mobile Phase: 0.2 mol/L Phosphate (pH 6.7)+0.05% NaN3
Flow rate: 0.8 mL/min
Detection: UV @ 280nm
Inj. volume: 10 μL
Sample: 1 Thyrobulobulin, 2 γ-Globulin,
3 Ovalbumin, 4 Ribonuclease A,
5 p-Aminobenzoic acid
TSKgel SuperSW mAb HR:
Good durability of performance for
protein sample injection
TOSOH BIOSCIENCE
Durability of TSKgel SuperSW mAb HR (3)
0
50
100
4 6 8 10 12 14
Elution time (min)
mV
1
Inj . 3
Rs=1.74
Inj . 303
Inj . 599
2
3
Rs=1.79
Rs=1.87
23
Column: TSKgel SuperSW mAb HR
(7.8 mm ID x 30 cm)
Mobile Phase: 0.2 mol/L Phosphate (pH 6.7)+0.05% NaN3
Flow rate: 0.8 mL/min
Detection: UV @ 280nm
Inj. volume: 10 μL
Sample: 1 γ-Globulin, 2 Cytochrome C,
3 DNP-L-Alanine
TSKgel SuperSW mAb HR:
Good durability of Rs for gamma-
globulin separation
TOSOH BIOSCIENCE 24
Conclusions
Features of new SEC columns:
• TSKgel SuperSW mAb HR exhibits superior resolving power for
IgG monomer and dimer compared to other SEC columns.
• TSKgel SuperSW mAb HTP exhibits equal separation between
IgG monomer and dimer in half the analysis time compared to the
current gold standard for mAb SEC - TSKgel G3000SWXL (5 μm
particle, 7.8 mm ID x 30 cm).
• TSKgel UltraSW Aggregate possesses a larger MW exclusion
limit and shows superior resolving power for multimers and
aggregates of large proteins, including thyroglobulin and IgG.
The performance of these columns was demonstrated by the
separation of IgG monomer, dimer, aggregates and fragment. The
columns show good lot-to-lot reproducibility and column durability.