This document summarizes an experiment investigating the robustness of the TOYOPEARL MX-Trp-650M resin for removing aggregates from monoclonal antibodies (mAbs) under different pH and conductivity conditions. It was found that the resin could remove aggregates to below the limit of detection across a range of pH from 4.4 to 5.6 and conductivities from 50 to 90 mS/cm. The resin showed particularly robust monomer recovery at pH 5.0 for all conductivities tested. Only deviations in pH greater than 0.6 or conductivity greater than 20 mS/cm from the optimal conditions resulted in potential process failure.

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TOYOPEARL MX-Trp-650M
Robustness in mAb Aggregate removal
August 2013
Judith Vajda, Egbert Müller

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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.

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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.

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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

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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

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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

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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

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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.