HPLC Analysis of an Oxidized Monoclonal Antibody Standard on BIOshell™ A400 Protein C4

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)
sample Native or oxidized mAb, 1 g/L, 0.05% trifluoroacetic acid
injection 1.0 µL
detector UV, 215 nm

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.
Categories Analytical Chromatography, Antibodies, Proteins
Featured Industry Life Science and Biopharma
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
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.
suitability application for HPLC

Materials

     
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