TOYOPEARL MX-Trp-650M is designed for efficient mixed-mode steps in protein purification: Multimodal cation exchanger High salt tolerance High binding capacity for IgG

1. TOSOH BIOSCIENCE
TOYOPEARL® MX-Trp-650M
A High Capacity Mixed-Mode Resin

2. 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

3. TOSOH BIOSCIENCE
Protein A mAb Purification
Capture Intermediate /Polishing
3

4. Tosoh Bioscience 4
TOYOPEARL MX-Trp-650M

5. 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

6. 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)

7. 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

8. 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

9. 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
HumanIgGDBC(g/L)
0.00 mol/L NaCl
0.10 mol/L NaCl
0.15 mol/L NaCl
0.20 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)

10. 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
DBC[mgmAb/mLresin]
NaCl concentration [mol/L]
Depending on the target molecule a certain conductivity even increases binding
capacity

11. 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

12. 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
DBC[mgmAb/mLresin]
pH

13. Tosoh Bioscience 13
Binding & Elution - Acidic Proteins
0
5
10
15
20
25
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
3
5
7
9
pH
ovalbumin
0
2
4
6
8
10
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
3
5
7
9
pH
Soybean trypsin inhibitor
0
5
10
15
20
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
3
5
7
9
pH
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

14. Tosoh Bioscience 14
Binding & Elution- Basic Proteins
0
20
40
60
80
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
3
5
7
9
pH
a-chymotrypsinoge A
0
20
40
60
80
100
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
3
5
7
9
pH
cytochrom C
0
20
40
60
80
0 20 40 60 80
Elution volume (mL)mAu(280nm)
3
5
7
9
pH
lysozyme
0
5
10
15
20
0 20 40 60 80
Elution volume (mL)
mAu(280nm)
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

15. 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

16. 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

17. 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)

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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)

24. 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

25. 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