UHPLC Analysis of NIST mAb Charge Variants: Effect of Particle and Ligand Morphology

UHPLC Analysis of NIST mAb Charge Variants


instrument Thermo Ultimate 3000 BioRS
column As indicated, 25 cm x 4.6 mm I.D., 5 µm
mobile phase   [A] 100 mM sodium phosphate, pH 7.0;
[B] 0.5 M sodium chloride in 100 mM sodium phosphate, pH 7.0
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.
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


Analysis Note Ion exchange chromatography (IEX) is a mode of chromatography that separates analytes based on charge.  This technique is employed in the biopharmaceutical industry to monitor for the presence of charge variants of a protein therapeutic, which usually result from post-translational modifications during expression.  However, not all IEX columns are the same, and not all weak or strong exchangers are the same as the underlying polymer particle can be different (more hydrophobic/hydrophilic) and can affect resolution of the variants.  This application demonstrates the importance of performing a column screen during IEX method development.  Among the three columns examined, the strong cation exchanger (SCX) exhibited better resolution of both acidic and basic charge variants from the main mAb peak.  The inset shows a zoomed in region of the chromatogram, clearly showing the improvements in resolution with the SCX column.
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Application No. MUR120618A


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