UHPLC Analysis NIST mAb Variants on Proteomix SCX-NP5: Comparing Salt and pH Gradients

UHPLC Analysis NIST mAb Variants on Proteomix SCX-NP5

Conditions

instrument Thermo Ultimate 3000 BioRS
column Proteomix SCX-NP5, 25 cm x 4.6 mm I.D., 5 µm
mobile phase Salt Gradient: [A] 100 mM sodium phosphate, pH 7.0; [B] 0.5 M sodium chloride in 100 mM sodium phosphate, pH 7.0;
pH Gradient: [A] 16 mM MES, 10 mM MOPS, 12 mM TAPS, 10 mM CAPSO, pH 5.6; [B] 10 mM MES, 12 mM MOPS, 14 mM TAPS, 16 mM CAPSO, pH 10.2; [C] B + 150 mM sodium chloride
gradient Salt Gradient: Hold at 10% B for 2 min; 10% B to 23% B in 30 min; 23% B to 100% B in 0.5 min; hold at 100% B for 5.5 min;
pH Gradient: 70% B to 90% B in 30 min; 90% B, 10% A to 100% C in 0.5 min; hold at 100% C for 5 min
flow rate 1.0 mL/min
column temp. 25 °C
detector UV, 280 nm
injection 6.0 µL
sample NIST mAb, 10 mg/mL, 12.5 mM histidine, pH 6.0

Description

Analysis Note Ion exchange chromatography (IEX) has become more prevalent in the biopharma industry resulting from the importance of monitoring and detecting charge variants.  IEX can be ran using one of two mobile phase "modes:" use of a salt gradient and use of a pH gradient.  These modes operate under different mechanisms: a salt gradient has the salt ions compete with analyte ions for binding to the stationary phase; a pH gradient changes the charge of both the stationary phase and the analytes, gradually causing the analytes to elute off the column as their interaction weakens.  This application demonstrates the importance of screening both a salt and pH gradient in IEX method development.  The pH gradient exhibited better separation of acidic variants whereas the salt gradient exhibited better separation of basic variants.
Legal Information Proteomix is a trademark of Sepax Technologies
LiChrosolv is a trademark of Merck KGaA, Darmstadt, Germany
Application No. MUR120618B

Materials

     
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