UHPLC Analyses of Reduced Infliximab on BIOshell(TM) IgG 1000 Ã… C4 and BIOshell A400 Protein C4

UHPLC Analyses of Reduced Infliximab on BIOshell™ IgG 1000 Ã… C4 and BIOshell A400 Protein C4

Conditions

column BIOshell A400 Protein C4, 10 cm x 2.1 mm I.D., 3.4 μm particles (66825-U)
column BIOshell IgG 1000 Ã… C4, 10 cm x 2.1 mm I.D., 2.7 μm particles (63288-U)
column temp. 75 °C
mobile phase [A] 70:30 water (0.1% TFA): acetonitrile (0.1% TFA); [B] 50:50 water (0.1% TFA): acetonitrile (0.1% TFA)
gradient 20% B to 50% B in 10 min; 50% B to 100% B in 5 min
flow rate 0.2 mL/min
pressure 1675 psi FPP (116 bar)
pressure 630 psi BIOshell A400 (43 bar)
pressure 1210 psi BIOshell lgG (83 bar)
sample reduced infliximab, 100 μg/mL, water (0.05% TFA)
injection 5 μL
detector UV, 215 nm

Description

Analysis Note Infliximab, due to the fact that it is a monoclonal antibody (mAb), will dissociate into a light chain (LC) and heavy chain (HC) upon disulfide bond reduction with a reducing agent. This application shows the advantages of using a wide pore, superficially porous particle (SPP)-packed column as opposed to a fully porous particle (FPP)-packed column or a smaller pore SPP-packed column. Better resolution of impurities and variants from the LC and HC is achieved with the BIOshell IgG column as well as enhanced retention of the LC and HC.
Featured Industry Pharmaceutical (small molecule)
Legal Information BIOshell is a trademark of Sigma-Aldrich Co. LLC
Other Notes Resuspended infliximab in 1 mL of reducing agent (0.1 M dithiobutylamine, 8 M guanidine hydrochloride, 0.1 M ammonium bicarbonate). Let reduction reaction occur for one hour at room temperature prior to analysis.
suitability application for UHPLC

Materials

     
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