Robustness in mAb Aggregate removal
Judith Vajda, Egbert Müller
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.
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.
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
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
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
• 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
• ∆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
• 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
• 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.