HPLC Analysis of an Oxidized Monoclonal Antibody Standard on BIOshell A400 Protein C4
CONDITIONS
column
BIOshell A400 Protein C4; 10 cm x 2.1 mm I.D., 3.4 μm particles (66825-U)
mobile phase
[A] 70:30 water (0.1% trifluoroacetic acid):acetonitrile (0.1% trifluoroacetic acid); [B] 50:50 water (0.1% trifluoroacetic acid): acetonitrile (0.1% trifluoroacetic acid)
gradient
0 to 50% B in 50 min; 50 to 100% B in 10 min
flow rate
0.2 mL/min
pressure
590 psi (41 bar)
detector
UV, 215 nm
injection
1.0 μL
sample
Native or oxidized mAb, 1 g/L, 0.05% trifluoroacetic acid
Description
Analysis Note
This application illustrates the analytical challenge in characterizing a heterogeneous monoclonal antibody standard. Once oxidized, this complexity significantly increases, as can be seen in the bottom panel of the figure. The BIOshell A400 Protein C4 column can provide partial resolution of the different oxidized species for qualitative identification of the oxidized variants.
Other Notes
The oxidized sample was prepared by re-suspending mAb in 500 μL water plus 500 μL 0.1% aqueous hydrogen peroxide. The reaction occurred for 24 hours at 10 °C prior to assay.
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