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.

1. TSKgel SW columns for mAb separation
TSKgel SuperSW mAb HR/HTP
TSKgel UltraSW Aggregate
TOSOH BIOSCIENCE

2. Structure of Antibodies
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
2

3. Therapeutic Proteins
• 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.
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3

4. Analysis of mAb Heterogeneity
Aggregation, dimer, fragmentation
>>> SEC
>>> HIC
Charge-related heterogeneity
Deamidation, termini modification etc.
Oxidation
>>> IEC
>>> HIC/RPC
Glycosylation
Full length mAb
Glycan composition
Glycation
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>>> IEC
>>> HILIC
>>> AFC/IEC
4

5. 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
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5

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
6

7. Characteristics of New Stationary Phases
Column
TSKgel 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
Silica gel
Functional group
Diol
Diol
Particle size
4 μm
3 μm
Pore size
25 nm
30 nm
10,000 - 500,000 Da
10,000 - 2,000,000 Da
Separation range
(for globular proteins)
Applications
Separation of mAb
monomer and dimer
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Fast separation of mAb
monomer and dimer
(UHPLC compatible).
Separation of mAb
aggregates.
7

8. Product line and specification
Analytical columns
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
Guardcolumns
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
TOSOH BIOSCIENCE
8

9. Chromatograms – Protein Standard
250
Columns:
4
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)
5
3
200
1
TSKgel
UltraSW Aggregate
(3μm, 30nm)
1'
2
2'
UV 280 nm
150
1
TSKgel
SuperSW mAb HR
(4μm, 25nm)
100
4
3
5
2
1'
2'
Vo
50
TSKgel
G3000SWXL
(5μm, 25nm)
0
0
2
4
Vo
6
8
Time [min]
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10
12
14
• 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
9

10. Calibration Curves - Protein Standard
7
TSKgel UltraSW Aggregate:
← Thyroglobulin trimer
← Thyroglobulin dimer
6
Covers separation range up to around 2
million Da. This implies better resolution
of aggregate/multimer of mAb.
← Thyroglobulin (MW 640,000)
← γ-Globulin (MW 155,000)
5
Log MW
← Ovalbulin (MW 47,000)
← Ribonuclease A
(MW 13,700)
4
TSKgel SuperSW mAb HR:
Exclusion limit (globular protein):
Shows a calibration curve similar to
TSKgel G3000SWXL and a shallower
slope than TSKgel UltraSW around Mw
range of gamma-globulin
2.5 x 106 Da (UltraSW Aggregate)
8.6 x 105 Da (SuperSW mAb HR)
3
2
p-Aminobenzoic acid
(MW 137) →
UltraSW Aggregate
SuperSW mAb HR
G3000SWXL
1
4
6
8
10
Elution time [min]
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12
14
10

11. 4
2
A. SuperSW
1
(1')
mAb HR
ABS @280nm
Chromatograms of Standard Proteins
5
3
(1') 1
5
4
3
H. SuperSW
mAb HTP
0
1
B. UltraSW
Aggregate
ABS @280 nm [AU]
2
2
3
4
5
6
7
Time [min]
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)
C. G3000SW XL
D. Brand A
(3 μm)
E. Brand B
(5 μm)
F. Brand C
(5 μm)
G. Brand D
(5 μm)
2
4
New TSKgel mAb SEC columns show superior
performance compared to competitor columns.
6
8
10
TOSOH BIOSCIENCE
Time [min]
12
14
11

12. UV 280 nm
Resolution of mAb Dimer and Monomer
monomer
A. SuperSW
mAb HR
dimer
Rs = 2.01
H. SuperSW
mAb HTP
2.0
Rs = 1.66
B. UltraSW
Aggregate
Rs = 1.69
Column:
UV 280 nm
C. G3000SW XL
Rs = 1.64
D. Brand A
(3 μm)
Rs = 1.95
E. Brand B
(5 μm)
Mobile phase:
Flow rate:
Temperature:
Detection:
Injection vol.:
Samples:
Rs = 1.56
YMC-Pack
F. Brand C Diol-300Rs = 1.59
(5 μm)
G. Brand D
(5 μm)
4
5
7
8
Time [min]
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3.0
3.5
Time [min]
4.0
4.5
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 G3000SW XL (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)
200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
A-G. 1.0 mL/min, H. 0.35 mL/min
25℃;
UV @ 280 nm
A-G. 10 μL H. 3.5 μL
IgG (human polyclonal) (A-G: 1.0 g/L, H: 4.5 g/L)
TSKgel SuperSW mAb HR:
Superior resolution of gamma-globulin dimer and monomer
Rs = 1.33
6
2.5
9
10
TSKgel SuperSW mAb HTP:
Good resolution of gamma-globulin dimer and monomer in
half of the analysis time of conventional columns
12

13. Separation of Therapeutic Antibody
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
Rs(3mer/2mer) =0.96
Rs(2mer/monomer) =2.32
TSKgel SuperSW mAb HR:
superior resolution of mAb dimer,
monomer and fragment
fragment
Rs(3mer/2mer) =1.32
Rs(2mer/monomer) =3.11
Df (mono./fragment) =0.095
TSKgel UltraSW Aggregate:
Superior resolution of mAb
trimer and dimer
Rs(3mer/2mer) =1.40
Rs(2mer/monomer) =2.89
TOSOH BIOSCIENCE
13

14. High Speed Separation of Therap. Antibody
120
TSKgel
SuperSW mAb HTP
(4.6mmID x 15cm)
100
Monoclonal antibody-2
UV280 nm
80
Flow rate: 0.50 mL/min
Pressure: 5.0 MPa
Rs(dimmer/monomer)=1.91
monomer
60
40
Flow rate: 0.35 mL/min
Pressure: 3.6 MPa
Rs(dimmer/monomer)=2.13
dimer
trimer
20
aggregates
0
0
Column:
Elution:
Flow rate:
Detection:
Sample:
1
2
3
4
Time [min]
TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
0.2 mol/L phosphate buffer (pH 6.7) + 0.05% NaN3
0.50 mL/min, 0.35 mL/min;
UV @ 280 n;m Temp.: 25℃;
5 uL monoclonal antibody-2 (mouse-human chimeric IgG, Erbitux)
TOSOH BIOSCIENCE
5
6
7
8
TSKgel SuperSW mAb HTP
showed superior resolution of mAb
dimer and monomer even at high flow
rate with low pressure drops
14

15. Separation of IgG Aggregates
250
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)
1
2
200
3
TSKgel
UltraSW Aggregate
(3μm, 30nm)
5
4
UV 280 nm
150
TSKgel
SuperSW mAb HR
(4μm, 25nm)
100
50
TSKgel
G3000SWXL
(5μm, 25nm)
TSKgel UltraSW Aggregate:
Wider separation window in
aggregate region (> trimer)
Vo
Vo
0
3
4
5
6
7
Time [min]
TOSOH BIOSCIENCE
8
9
10
15

16. Effect of Flow Rate on Multimer Separation
50
50
1.0 mL/min
UV 280 nm
30
30
tetramer
20
30
20
10
20
10
0
6
8
10
0
10
15
Time [min]
Column:
Mobile phase :
Flow rate:
Temperature:
Detection:
Injection vol.:
Sample:
TSKgel UltraSW Aggregate (7.8 mm ID x 30 cm)
200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
0.2~1.0 mL/min
25℃
UV @ 280 nm
10 μL
IgG (human monoclonal, MAb-02, 5 g/L)
TOSOH BIOSCIENCE
pentamer
10
0
4
0.2 mL/min
40
UV 280 nm
40
UV 280 nm
40
50
0.4 mL/min
20
Time [min]
25
20
30
40
50
Time [min]
Pentamer was separated at lower flow rate,
< 0.4 mL/min on UltraSW Aggregate
16

17. Effect of Flow Rate on Separation
Theoretical plates and flow rate
Resolution and flow rate
6
40,000
35,000
5
30,000
4
dimer / monomer
Rs
TP
25,000
20,000
3
monomer
15,000
2
10,000
trimer / dimer
dimer
5,000
1
trimer
tetramer
tetramer / trimer
0
0
0.0
0.2
0.4
0.6
0.8
Flow rate [mL/min]
1.0
1.2
0.0
0.2
0.4
0.6
0.8
Flow rate [mL/min]
1.0
1.2
The lower the flow rate, the higher the column performance
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17

18. Effect of Flow Rate - TSKgel SuperSW mAb HR
van Deemter plot
Resolution (IgG dimer and monomer)
250
Thyroglobulin
2.4
150
55
2.5
200
60
2.3
45
γ-Globulin
HETP [um]
40
100
35
50
30
25
0
Ovalbumin
20
-50
Ribonuclease A
15
-100
10
2.2
2.1
2.0
1.9
1.7
-150
p-Aminobenzoic acid
0.5
1.0
1.5
2.0
u (Linear velocity) [cm/min]
1.0 mL/min
1.6
1.5
2.5
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)
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0.5 mL/min
1.8
-200
5
0
0.0
Rs (IgG dimer/monomer)
50
0.0
0.5
1.0
1.5
2.0
u (Linear velocity) [cm/min]
2.5
Sample: IgG (human polyclonal) (4.5 g/L)
Other conditions were the same as
described left.
18

19. Effect of Flow Rate - TSKgel SuperSW mAb HTP
Resolution (IgG dimer and monomer)
Pressure drops
6
1.8
1.6
Standard flow rate
1.4
Rs =1.51
(0.476 mL/min)
1.2
Pressure Drop [MPa]
7
2.0
Rs (IgG dimer/monomer)
2.2
5.6 MPa
5
4
3
Standard flow rate
2
1
0
1.0
0.0
0.1
0.2
0.3
0.4
Flow rate [mL/min]
0.5
High speed separation
Complete separation of IgG
dimer and monomer even with
high flow rate
0.0
0.2
0.3
0.4
Flow rate [mL/min]
0.5
Low pressure drops
Compatible to both UHPLC and
conventional HPLC
Column:
Mobile phase :
Flow rate:
Detection:
Samples:
TOSOH BIOSCIENCE
0.1
TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm)
200 mmol/L phosphate buffer (pH 6.7) + 0.05% NaN3
0.143~0.476 mL/min;
Temperature: 25℃
UV @ 280 nm,
Injection vol.: 5 μL
IgG (human polyclonal) (4.5 g/L)
19

20. Lot to Lot Reproducibility
250
5
4
1
TSKgel SuperSW mAb HR
exhibited good gel lot to lot
reproducibility
3
2
200
Lot. 503T
mV
150
Lot. 502T
100
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
50
Lot. 501T
0
4
6
8
10
12
14
16
18
Elution time (min)
TOSOH BIOSCIENCE
20

21. Durability of TSKgel SuperSW mAb HR (1)
Change of retention time and theoretical plates for p-ABA
100
90
90
TP (%)
110
100
RT(%)
110
80
80
70
70
60
60
50
50
0
200
400
Inj. #
Column:
Eluent:
Flow rate:
Sample:
600
0
200
400
600
Inj. #
TSKgel SuperSW mAb HR (7.8 mm ID x 30 cm)
0.2 mol/L Phosphate Buffer (pH 6.7)+0.05% NaN3
0.8 mL/min Detection: UV280nm Inj. volume: 10 μL
1 Thyrobulobulin, 2 γ-Globulin, 3 Ovalbumin, 4 Ribonuclease A, 5 p-Aminobenzoic acid
TOSOH BIOSCIENCE
21

22. Durability of TSKgel SuperSW mAb HR (2)
TSKgel SuperSW mAb HR:
Good durability of performance for
protein sample injection
5
4
150
1
3
2
Inj. 561
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
mV
100
Inj. 301
50
Inj. 1
0
4
6
8
10
12
14
Elution time (min)
TOSOH BIOSCIENCE
22

23. Durability of TSKgel SuperSW mAb HR (3)
100
2
TSKgel SuperSW mAb HR:
Good durability of Rs for gammaglobulin separation
3
1
Rs=1.74
mV
Inj. 599
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
50
Rs=1.79
Inj. 303
Rs=1.87
Inj. 3
0
4
6
8
10
12
14
Elution time (min)
TOSOH BIOSCIENCE
23

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.
TOSOH BIOSCIENCE
24