TOSOH BIOSCIENCE

TOYOPEARL® MX-Trp-650M

A High Capacity Mixed-Mode Resin

Tosoh Bioscience

Why Use Mixed Mode

• Direct load of high conductivity feedstocks

• Additional chromatographic mode with unique

selectivity that combines hydrophobic and ion

exchange characteristics

• Selectivity can be modulated by salt and pH

variation

• Suitable for aggregate removal in

intermediate/polishing steps

2

TOSOH BIOSCIENCE

Protein A mAb Purification

Capture Intermediate /Polishing

3

Tosoh Bioscience 4

TOYOPEARL MX-Trp-650M

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

5

Tosoh Bioscience 6

High binding capacity for IgG

Column: TOYOPEARL MX-Trp-650M (6 mm ID x 4 cm)

Loading buffer: 0.05 mol/L sodium acetate buffer (pH 4.7) + 0.1 mol/L sodium chloride

Flow rate: 212, 425, 637, 849 cm/h

Detection: UV @ 280 nm

Temperature: ambient

Sample: polyclonal human IgG (1 mg/mL)

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

7

Tosoh Bioscience 8

High DBC at High Conductivity

Resin Mean

Particle Size

DBC

@12 mS/cm

Recovery

@12 mS/cm

DBC

@17 mS/cm

Recovery

@17 mS/cm

TOYOPEARL MX-Trp 75 µm 95 g/L 97 % 48 g/L 96 %

Brand M (Agarose) 75 µm 14 g/L 86 % 11 g/L 85 %

Toyopearl MX-Trp-650M or Brand M agarose based multimodal CEX

Column size: 6 mm ID x 4 cm

Binding Buffer: 50 mmol/L acetate buffer (pH 4.7) + 100 mmol/L NaCl (conductivity 12 mS/cm) or

50 mmol/L acetate buffer (pH 4.7) + 150 mmol/L NaCl (conductivity 17 mS/cm)

Flow rate: 212 cm/h; Detection: UV @ 280 nm;

Sample: polyclonal IgG

DBC measured at 10 % breakthrough

Recovery and binding capacity of TOYOPEARL MX-Trp-650M at high conductivity is

much higher than those of other commercial multimodal CEX resins

Tosoh Bioscience 9

Effect of Buffer pH and Salt on DBC

0

20

40

60

80

100

120

3.5 4 4.5 5 5.5 6 6.5

Loading buffer pH

Hu

ma

n I

gG

DB

C (

g/L

)

0.00 mol/L NaCl0.10 mol/L NaCl

0.15 mol/L NaCl0.20 mol/L NaCl

Dynamic binding capacities (DBC) were determined at 10% breakthrough.

Column: 6 mm ID x 4 cm

Binding Buffer 1: 0.05 mol/L acetate buffer (pH 4.0 - 6.0) + 0 - 0.2 mol/L NaCl

Flow rate: 1.0 mL/min (212 cm/h)

Detection: UV @ 280 nm

Sample: human polyclonal IgG (1 mg/mL)

Tosoh Bioscience 10

Effect of Salt Concentration on DBC

Dynamic binding capacities (DBC) were determined at 10% breakthrough.

Column:

Binding Buffer: 0.1 mol/L acetate buffer (pH 4.25) + 0 - 0.35 mol/L NaCl

Flow rate:

Detection: UV @ 280 nm

Sample: humanized monoclonal IgG

0

10

20

30

40

50

60

70

80

90

100

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

DB

C [

mg

mA

b /

mL

resin

]

NaCl concentration [mol/L]

Depending on the target molecule a certain conductivity even increases binding

capacity

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

11

Tosoh Bioscience 12

Effect of Buffer pH on DBC

Dynamic binding capacities (DBC) were determined at 10% breakthrough.

Column: 6.6 mm ID x 2.2 cm

Binding Buffer 0.1 mol/L acetate buffer (pH 4.0 - 6.0) + 0.2 mol/L NaCl

Linear Velocity: 150 cm/h

Detection: UV @ 280 nm

Sample: humanized monoclonal IgG

0

10

20

30

40

50

60

70

80

90

100

3 3.5 4 4.5 5 5.5

DB

C [

mg

mA

b /

mL

resin

]

pH

Tosoh Bioscience 13

Binding & Elution - Acidic Proteins

0

5

10

15

20

25

0 20 40 60 80

Elution volume (mL)

mA

u (

280 n

m)

3

5

7

9

pH

ovalbumin

0

2

4

6

8

10

0 20 40 60 80

Elution volume (mL)

mAu (

280 n

m)

3

5

7

9

pH

Soybean trypsin inhibitor

0

5

10

15

20

0 20 40 60 80

Elution volume (mL)

mA

u (

280 n

m)

3

5

7

9pH

BSA

Column: TOYOPEARL MX-Trp-650M (5 mm ID X 5 cm)

Buffer A: 0.02 mol/L Acetic acid, MES and HEPES

containing 0.15 mol/L NaCl (pH 4)

Buffer B: 0.02 mol/L Acetic acid, MES and HEPES

containing 0.15 mol/L NaCl (pH 8)

Gradient: 60 min linear from buffer A to buffer B

Flow-rate: 0.5 mL/min

Detection: UV @ 280 nm

Protein load: 1 mg

Tosoh Bioscience 14

Binding & Elution- Basic Proteins

0

20

40

60

80

0 20 40 60 80

Elution volume (mL)

mA

u (

280 n

m)

3

5

7

9

pH

a-chymotrypsinoge A

0

20

40

60

80

100

0 20 40 60 80

Elution volume (mL)

mA

u (

280 n

m)

3

5

7

9

pH

cytochrom C

0

20

40

60

80

0 20 40 60 80

Elution volume (mL)m

Au (

280 n

m)

3

5

7

9

pH

lysozyme

0

5

10

15

20

0 20 40 60 80

Elution volume (mL)

mA

u (

280 n

m)

3

5

7

9

pH

ribonuclease A

Column: TOYOPEARL MX-Trp-650M (5 mm ID X 5 cm)

Buffer A: 0.02 mol/L Acetic acid, MES and HEPES containing 0.15 mol/L NaCl (pH 4)

Buffer B: 0.02 mol/L Acetic acid, MES and HEPES containing 0.15 mol/L NaCl (pH 8)

Gradient: 60 min linear from buffer A to buffer B

Flow-rate: 0.5 mL/min, Detection: UV @ 280 nm, Protein load: 1 mg

Gradient elution with both salt and pH gradient will further sharpen peaks

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

15

Tosoh Bioscience 16

Good Pressure Flow Properties

Back pressure of TOYOPEARL MX-Trp-650M column is less than half of other

commercial multimodal CEX resins

Resin: TOYOPEARL MX-Trp-650M (red) Agarose based multimodal CEX gel (blue)

Column size: 22 mm ID x 20 cm

Eluent: distilled water

Tosoh Bioscience 17

High DBC at High Flow Rates

Column: TOYOPEARL MX-Trp-650M (6 mm ID x 4 cm)

Loading buffer: 0.05 mol/L sodium acetate buffer (pH 4.7) + 0.1 mol/L sodium chloride

Flow rate: 212, 425, 637, 849 cm/h

Detection: UV @ 280 nm

Temperature: ambient

Sample: polyclonal human IgG (1 mg/mL)

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

18

Tosoh Bioscience 19

Sharper Peaks – Less Peak Tailing

Resins: TOYOPEARL MX-Trp-650M, Brand M;

Column size: 7.5 mm ID x 7.5 cm

Mobile phase: Buffer A: 20 mmol/L phosphate (pH 7.0); Buffer B: 20 mmol/L phosphate

+ 1.0 mol/L NaCl (pH 7.0)

Gradient: 30 min linear from buffer A to buffer B

Flow rate: 1.0 mL/min

Detection: UV @ 280nm

Sample: Trypsinogen (6.6 mg/mL); cytochrome C (3.6 mg/mL); lysozyme (6.6 mg/mL);

Sample vol.: 25 μL

Tosoh Bioscience 20

Low Pool Volume Elution Peak

Column: TOYOPEARL MX-Trp-650M (6 mm ID x 4 cm)

Binding buffer; 0.05 mol/L acetate buffer (pH 4.7) + 0.1 mol/L NaCl

Elution buffer: 0.1 mol/L Tris-HCl buffer (pH 8.5) + 0.3 mol/L NaCl

Adsorption flow rate: 1.0 mL/min (212 cm/h)

Elution flow rate: 2.0 mL/min (started at 124 min)

Detection: UV @ 280 nm

Sample: CHO cell culture media, monoclonal antibody (1 mg/mL) diluted with binding buffer

Tosoh Bioscience 21

Purified mAb Fraction Aggregate content of 0.9 % after TOYOPEARL rProtein A capture and MX-Trp polishing

Column: TSKgel G3000SWXL (7.8 mm ID X 30 cm)

Mobile Phase: 0.1 mol/L phosphate buffer (pH 6.7) + 0.1 mol/L Na2SO4

Flow rate: 1.0 mL/min

Detection: UV @ 280 nm

Sample: Monoclonal antibody fraction eluted from TOYOPEARL MX-Trp-650M

@ 100 mM acetate pH 5.5 + 0.4 mol/L NaCl

Injection Vol.: 20 µL

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Properties

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• High binding capacity for antibodies

• High DBC at higher feedstock conductivities

• Strong influence of pH on adsorption and elution

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

22

Tosoh Bioscience 23

Alkaline Stability (CIP)

Alkaline cleaning (CIP) conditions

Alkaline solution: 0.5 mol/L NaOH; 3CV

Buffer solution: 0.1 mol/L Tris-HCl pH 8.5 + 0.3 mol/L NaCl; 5CV

Flow rate: 1 mL/min.

DBC Measurement

Column: TOYOPEARL MX-Trp-650M (6 mm ID X 4 cm)

Binding buffer: 0.05 mol/L acetate buffer (pH 4.3) + 0.1 mol/L NaCl;

Flow rate: 1 mL/min; Detection: UV @ 280 nm

Sample: polyclonal human IgG (1 mg/mL)

Tosoh Bioscience

TOYOPEARL MX-Trp-650M Summary

• Multimodal cation exchanger offering

hydrophobic and ionic interactions

• Binding capacity for antibodies almost reaches

that of grafted ion exchange resins

• High DBC at higher conductivities

• pH is a powerful tool for optimization

• Can be run at fast linear velocities to increase

throughput

• Excellent binding and elution kinetics reduce

process pool volumes

• Maintains high capacity after many CIP cycles

24

Tosoh Bioscience 25

TOYOPEARL MX-Trp-650M

Product P/N Volume

TOYOPEARL MX-Trp-650M 22817 25 mL

TOYOPEARL MX-Trp-650M 22818 100 mL

TOYOPEARL MX-Trp-650M 22819 1000 mL

ToyoScreen MX-Trp-650M 22824 1 mL x 6

ToyoScreen MX-Trp-650M 22825 5 mL x 6