tskgel sw columns for mab separation

24
TOSOH BIOSCIENCE TSKgel SW columns for mAb separation TSKgel SuperSW mAb HR/HTP TSKgel UltraSW Aggregate

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Page 1: TSKgel SW columns for mAb separation

TOSOH BIOSCIENCE

TSKgel SW columns for mAb separation

TSKgel SuperSW mAb HR/HTP

TSKgel UltraSW Aggregate

Page 2: TSKgel SW columns for mAb separation

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.

Page 3: TSKgel SW columns for mAb separation

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

Page 4: TSKgel SW columns for mAb separation

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

Page 5: TSKgel SW columns for mAb separation

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

Page 6: TSKgel SW columns for mAb separation

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)

Page 7: TSKgel SW columns for mAb separation

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

Page 8: TSKgel SW columns for mAb separation

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

Page 9: TSKgel SW columns for mAb separation

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

Page 10: TSKgel SW columns for mAb separation

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) →

Page 11: TSKgel SW columns for mAb separation

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)

Page 12: TSKgel SW columns for mAb separation

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

Page 13: TSKgel SW columns for mAb separation

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

Page 14: TSKgel SW columns for mAb separation

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

Page 15: TSKgel SW columns for mAb separation

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)

Page 16: TSKgel SW columns for mAb separation

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)

Page 17: TSKgel SW columns for mAb separation

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

Page 18: TSKgel SW columns for mAb separation

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

Page 19: TSKgel SW columns for mAb separation

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

Page 20: TSKgel SW columns for mAb separation

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

Page 21: TSKgel SW columns for mAb separation

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

Page 22: TSKgel SW columns for mAb separation

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

Page 23: TSKgel SW columns for mAb separation

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

Page 24: TSKgel SW columns for mAb 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.