TOYOPEARL MX-Trp-650M

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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

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TOYOPEARL MX-Trp-650M

  1. 1. TOSOH BIOSCIENCETOYOPEARL® MX-Trp-650MA High Capacity Mixed-Mode Resin
  2. 2. Tosoh BioscienceWhy Use Mixed Mode• Direct load of high conductivity feedstocks• Additional chromatographic mode with uniqueselectivity that combines hydrophobic and ionexchange characteristics• Selectivity can be modulated by salt and pHvariation• Suitable for aggregate removal inintermediate/polishing steps2
  3. 3. TOSOH BIOSCIENCEProtein A mAb PurificationCapture Intermediate /Polishing3
  4. 4. Tosoh Bioscience 4TOYOPEARL MX-Trp-650M
  5. 5. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles5
  6. 6. Tosoh Bioscience 6High binding capacity for IgGColumn: 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 chlorideFlow rate: 212, 425, 637, 849 cm/hDetection: UV @ 280 nmTemperature: ambientSample: polyclonal human IgG (1 mg/mL)
  7. 7. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles7
  8. 8. Tosoh Bioscience 8High DBC at High ConductivityResin MeanParticle SizeDBC@12 mS/cmRecovery@12 mS/cmDBC@17 mS/cmRecovery@17 mS/cmTOYOPEARL 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 CEXColumn size: 6 mm ID x 4 cmBinding Buffer: 50 mmol/L acetate buffer (pH 4.7) + 100 mmol/L NaCl (conductivity 12 mS/cm) or50 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 IgGDBC measured at 10 % breakthroughRecovery and binding capacity of TOYOPEARL MX-Trp-650M at high conductivity ismuch higher than those of other commercial multimodal CEX resins
  9. 9. Tosoh Bioscience 9Effect of Buffer pH and Salt on DBC0204060801001203.5 4 4.5 5 5.5 6 6.5Loading buffer pHHumanIgGDBC(g/L)0.00 mol/L NaCl0.10 mol/L NaCl0.15 mol/L NaCl0.20 mol/L NaClDynamic binding capacities (DBC) were determined at 10% breakthrough.Column: 6 mm ID x 4 cmBinding Buffer 1: 0.05 mol/L acetate buffer (pH 4.0 - 6.0) + 0 - 0.2 mol/L NaClFlow rate: 1.0 mL/min (212 cm/h)Detection: UV @ 280 nmSample: human polyclonal IgG (1 mg/mL)
  10. 10. Tosoh Bioscience 10Effect of Salt Concentration on DBCDynamic 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 NaClFlow rate:Detection: UV @ 280 nmSample: humanized monoclonal IgG01020304050607080901000 0.05 0.1 0.15 0.2 0.25 0.3 0.35DBC[mgmAb/mLresin]NaCl concentration [mol/L]Depending on the target molecule a certain conductivity even increases bindingcapacity
  11. 11. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles11
  12. 12. Tosoh Bioscience 12Effect of Buffer pH on DBCDynamic binding capacities (DBC) were determined at 10% breakthrough.Column: 6.6 mm ID x 2.2 cmBinding Buffer 0.1 mol/L acetate buffer (pH 4.0 - 6.0) + 0.2 mol/L NaClLinear Velocity: 150 cm/hDetection: UV @ 280 nmSample: humanized monoclonal IgG01020304050607080901003 3.5 4 4.5 5 5.5DBC[mgmAb/mLresin]pH
  13. 13. Tosoh Bioscience 13Binding & Elution - Acidic Proteins05101520250 20 40 60 80Elution volume (mL)mAu(280nm)3579pHovalbumin02468100 20 40 60 80Elution volume (mL)mAu(280nm)3579pHSoybean trypsin inhibitor051015200 20 40 60 80Elution volume (mL)mAu(280nm)3579pHBSAColumn: TOYOPEARL MX-Trp-650M (5 mm ID X 5 cm)Buffer A: 0.02 mol/L Acetic acid, MES and HEPEScontaining 0.15 mol/L NaCl (pH 4)Buffer B: 0.02 mol/L Acetic acid, MES and HEPEScontaining 0.15 mol/L NaCl (pH 8)Gradient: 60 min linear from buffer A to buffer BFlow-rate: 0.5 mL/minDetection: UV @ 280 nmProtein load: 1 mg
  14. 14. Tosoh Bioscience 14Binding & Elution- Basic Proteins0204060800 20 40 60 80Elution volume (mL)mAu(280nm)3579pHa-chymotrypsinoge A0204060801000 20 40 60 80Elution volume (mL)mAu(280nm)3579pHcytochrom C0204060800 20 40 60 80Elution volume (mL)mAu(280nm)3579pHlysozyme051015200 20 40 60 80Elution volume (mL)mAu(280nm)3579pHribonuclease AColumn: 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 BFlow-rate: 0.5 mL/min, Detection: UV @ 280 nm, Protein load: 1 mgGradient elution with both salt and pH gradient will further sharpen peaks
  15. 15. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles15
  16. 16. Tosoh Bioscience 16Good Pressure Flow PropertiesBack pressure of TOYOPEARL MX-Trp-650M column is less than half of othercommercial multimodal CEX resinsResin: TOYOPEARL MX-Trp-650M (red) Agarose based multimodal CEX gel (blue)Column size: 22 mm ID x 20 cmEluent: distilled water
  17. 17. Tosoh Bioscience 17High DBC at High Flow RatesColumn: 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 chlorideFlow rate: 212, 425, 637, 849 cm/hDetection: UV @ 280 nmTemperature: ambientSample: polyclonal human IgG (1 mg/mL)
  18. 18. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles18
  19. 19. Tosoh Bioscience 19Sharper Peaks – Less Peak TailingResins: TOYOPEARL MX-Trp-650M, Brand M;Column size: 7.5 mm ID x 7.5 cmMobile 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 BFlow rate: 1.0 mL/minDetection: UV @ 280nmSample: Trypsinogen (6.6 mg/mL); cytochrome C (3.6 mg/mL); lysozyme (6.6 mg/mL);Sample vol.: 25 μL
  20. 20. Tosoh Bioscience 20Low Pool Volume Elution PeakColumn: 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 NaClElution buffer: 0.1 mol/L Tris-HCl buffer (pH 8.5) + 0.3 mol/L NaClAdsorption flow rate: 1.0 mL/min (212 cm/h)Elution flow rate: 2.0 mL/min (started at 124 min)Detection: UV @ 280 nmSample: CHO cell culture media, monoclonal antibody (1 mg/mL) diluted with binding buffer
  21. 21. Tosoh Bioscience 21Purified mAb FractionAggregate content of 0.9 % after TOYOPEARL rProtein A capture and MX-Trp polishingColumn: TSKgel G3000SWXL (7.8 mm ID X 30 cm)Mobile Phase: 0.1 mol/L phosphate buffer (pH 6.7) + 0.1 mol/L Na2SO4Flow rate: 1.0 mL/minDetection: UV @ 280 nmSample: Monoclonal antibody fraction eluted from TOYOPEARL MX-Trp-650M@ 100 mM acetate pH 5.5 + 0.4 mol/L NaClInjection Vol.: 20 µL
  22. 22. Tosoh BioscienceTOYOPEARL MX-Trp-650M Properties• Multimodal cation exchanger offeringhydrophobic 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles22
  23. 23. Tosoh Bioscience 23Alkaline Stability (CIP)Alkaline cleaning (CIP) conditionsAlkaline solution: 0.5 mol/L NaOH; 3CVBuffer solution: 0.1 mol/L Tris-HCl pH 8.5 + 0.3 mol/L NaCl; 5CVFlow rate: 1 mL/min.DBC MeasurementColumn: 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 nmSample: polyclonal human IgG (1 mg/mL)
  24. 24. Tosoh BioscienceTOYOPEARL MX-Trp-650M Summary• Multimodal cation exchanger offeringhydrophobic and ionic interactions• Binding capacity for antibodies almost reachesthat 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 increasethroughput• Excellent binding and elution kinetics reduceprocess pool volumes• Maintains high capacity after many CIP cycles24
  25. 25. Tosoh Bioscience 25TOYOPEARL MX-Trp-650MProduct P/N VolumeTOYOPEARL MX-Trp-650M 22817 25 mLTOYOPEARL MX-Trp-650M 22818 100 mLTOYOPEARL MX-Trp-650M 22819 1000 mLToyoScreen MX-Trp-650M 22824 1 mL x 6ToyoScreen MX-Trp-650M 22825 5 mL x 6

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