ProteinSimple eBook - 12

Simplifying Protein Analysis * Mastering Charge Heterogeneity Analysis of Therapeutic Proteins

into very concentrated sample zones. Under these conditions, hydrophobic
regions may aggregate or interact, which can in turn affect the resolution and
reproducibility of a charge heterogeneity proﬁle. Addition of solubilizers such
as urea into the sample eliminates aggregation effectively and improves
separation as shown in Figure 2B.
After a protein's peak proﬁle has been stabilized, resolution can be
addressed by adding narrow pH range ampholytes to the sample matrix. In
Figure 2C1 the addition of narrow-range ampholytes results in near-baseline
resolution of all isoforms. Triplicate run overlays shown in Figure 2C2
demonstrate the separation is very reproducible while providing high resolution
of 0.04 pH units. The complete icIEF method development process, from
compound screening (Figure 1) to obtaining a ﬁnal analytical method
(Figure 2C), was completed in only 2.5 hours.
Once developed, an analytical method can be further optimized for robustness
by implementing computational tools such as Central Composite Design of
Experiment. A step-by-step description of executing a DOE for iCE3 method
ﬁne-tuning and characterizing is available online at www.proteinsimple.com.
Considerations for Method Robustness
Sample components, especially salts, can compromise the resolution and
robustness of icIEF methods. During the focusing process, ions that do not
have a zwitterionic or neutral charge state are driven out of the capillary by
electrokinesis. As these charged compounds leave the capillary, they are
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C1
C2

Figure 2. The complete icIEF method development process-from the screening
of compounds in a pH gradient (A), to the addition of solubilizers (B), to the
addition of narrow-range ampholytes (C)-can be completed in 2.5 hours.

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