What is the recommended method to analyze lowly abundant antibody charged variants? - WKB106881

ENVIRONMENT

• Antibody charged variant
• Separation methods
• IEX columns
• BioResolve SCX mAb

ANSWER

Ion exchange using the BioResolve SCX mAb column with fluorescence detection would separate the low-concentration antibody charged variants, and fluorescence detection would give approximately 10 times greater sensitivity than UV absorbance at 280 nm.

ADDITIONAL INFORMATION

Preconcentration with a molecular weight cutoff (MWCO) filter (10K for IgG size protein) is a good approach, more forgiving and less prone to speciation of proteins. The MWCO filter technique would still necessitate investigating the best injection volume or pre-diluting the concentrated sample to reduce pH and ionic strength. 

Larger injection volumes are an option on IEX columns, but a sample in PBS will need to be diluted 3X or 4X in water (or 10mM MES at low pH—roughly 5.5-6) prior to loading on the column.

These measures are necessary to ensure that lowly abundant charge variants are analyzed more reproducibly (the charged variants can be 5% lower than the protein concentration, which might start at 10 µg/mL).