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Robustness toyopearl mx trp650 m

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  • 1. TOSOH BIOSCIENCE TOYOPEARL MX-Trp-650M Robustness in mAb Aggregate removal August 2013 Judith Vajda, Egbert Müller
  • 2. TOSOH BIOSCIENCE Materials & Methods • TOYOPEARL MX-Trp-650M • TSKgel G3000SWxl • Example mAb • Heat induced aggregation was enforced by incubation at 75 °C for 5 min Quantitative analysis via SEC • Human γ-globulin • DBC10: 1 g/l protein solution was loaded applying 150 cm/h. 100 mM acetate buffer was set to the corresponding pHs, conductivity was adjusted with sodium chloride. Columns: 6.6 mm ID x 2.1 ml L. • Aggregate removal experiments: 4 g aggregated antibody per ml resin were loaded, flow: 150 cm/h, load: 100 mM acetate, pH 5.0, 20 mS/cm, elute: 100 mM acetate, pH and conductivity as will be mentioned. Conductivity was adjusted by adding sodium chloride. A linear gradient from 0-100 % B over 50 CV was applied. Yields were calculated by AUC. • The goal of the study was to investigate the extent of the operating frame of MX-Trp-650M for an example application.
  • 3. TOSOH BIOSCIENCE DBC10 for the example mAb and human γ-globulin mAb optimum (> 70 mg/ml): pH 5.0 and 17.5 mS/cm. > 60 mg/ml are still achieved for ∆pH < 0.2 and ∆conductivity 5 mS/cm. Human γ-globulin optimum (70 mg/ml): pH 4.5 and 10 mS/cm. 60 mg/ml are still achieved for ∆pH < 0.2 and ∆conductivity 5 mS/cm.
  • 4. TOSOH BIOSCIENCE Aggregated mAb sample - SEC 100 µl aggregated mAb sample before TOYOPEARL MX-Trp-650M 0.1 M sodium phosphate buffer + 0.1 M sodium sulfate, pH 6.7 1 ml/min, TSKgel G3000SWxl ~10 % aggregates 100 µl elution pool pH 5.0, 70 mS/cm after TOYOPEARL MX-Trp-650M 0.1 M sodium phosphate buffer + 0.1 M sodium sulfate, pH 6.7 1 ml/min, TSKgel G3000SWxl aggregates < LOD
  • 5. TOSOH BIOSCIENCE Resolution of mAb monomer and aggregates – pH 4.4 50 mS/cm: no recovery 70 mS/cm: 83 % monomer yield 90 mS/cm: 83 % monomer yield, More concentrated elution pH 4.4 50 mS/cm
  • 6. TOSOH BIOSCIENCE Resolution of mAb monomer and aggregates – pH 5.0 50 mS/cm: 90 % monomer yield 70 mS/cm: 93 % monomer yield 90 mS/cm: 94 % monomer yield
  • 7. TOSOH BIOSCIENCE Resolution of mAb monomer and aggregates – pH 5.6 50 mS/cm: 84 % monomer yield Rs 1.8 70 mS/cm: 91 % monomer yield Rs 1.6 90 mS/cm: 89 % monomer yield Rs 1.2
  • 8. TOSOH BIOSCIENCE Conclusions • The maximum DBC10 for human γ-globulin and the example mAb vary with regards to pH and conductivity. Low conductivity is preferrable for human γ- globulin, while it seems like a medium conductivity increases DBC for the mAb. • ∆conductivities of about 5 mS/cm and ∆pH of 0.2 will still achieve capacities amounting 85-90 % of the maximum capacity. The capacity loss depends on both parameters. • Considering a step gradient to be applicable for Rs > 1.5, aggregate removal by a step gradient would be succesful for all the tested conditions except for pH 4.4 and 50 mS/cm, as well as pH 5.6 and 90 mS/cm. only ∆pH 0.6 and ∆conductivity 20 mS/cm will lead to process failure if directing either both towards accelerated elution or reduced elution. • pH 5.0 shows robust monomer elution for all applied conductivities, while aggregates remain efficiently adsorbed to the resin. • Conductivity required for elution decreases by ~ 5 mS/cm for every of the tested pH-steps in increasing order.

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