ProteinSimple eBook - 13

Simplifying Protein Analysis * Mastering Charge Heterogeneity Analysis of Therapeutic Proteins

Figure 3. The separation of IgG 1 Kappa demonstrates the
adverse effects of salts (A). Triplicate runs at 100 mM NaCl show
that salt also affects reproducibility (B).

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replaced by the anolyte's hydronium and catholyte's hydroxyl ions to
maintain electroneutrality. This results in a high separation current along
with compression of the pH gradient.
The separation of IgG 1 Kappa in Figure 3A clearly demonstrates salt's
adverse effects on icIEF analysis. The resulting pH gradient compression can
be observed by both the loss in resolution of IgG 1 Kappa charge isoforms
and the pH shift of the pI 9.46 marker.
Replicate runs at the highest salt concentration shown in Figure 3B
illustrate the combined impact of salt-related high separation current and
gradient compression on IgG 1 Kappa charge isoforms. The distribution of
IgG 1 Kappa charge variants migrates toward lower pH, and forms an
unresolved mound as it either degrades and/or aggregates in this extreme
separation environment.
Separation artifacts due to high salt concentration can be easily avoided
by reducing the concentration of salt components in the sample prior to
analysis. In the case of formulations with high protein concentration, the
act of diluting the protein down to the ﬁnal working concentration in sample
solution, typically in the range of 200-250 µg/mL for a mAb, will eliminate
enough ionic strength to allow for successful iCE analysis. For formulations
with low protein concentrations (<10× dilution to ﬁnal sample concentration),
a buffer exchange step may be needed to achieve best results.
As with all separation techniques, high-quality reagents should be used

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