Robustness toyopearl mx trp650 m

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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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DBC10for 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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Rs 1.8


Rs 1.6


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.

Robustness toyopearl mx trp650 m

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