icIEF methods require optimization of only a few parameters. The first step in method development is to screen new compounds with a generic method employing a Pharmalyte 3–10 pH gradient as shown in Figure 1A. For many molecules, methods with this broad pH range provide sufficient performance and do not require further development. For more challenging molecules with complex peak profiles and/or limited solubility, method optimization can be accomplished using the following simple strategies.
In icIEF, proteins simultaneously lose surface charge while being focused 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 1B.
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 1C1 the addition of narrow-range ampholytes results in near-baseline resolution of all isoforms. Triplicate run overlays shown in Figure 1C2 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 1A) to obtaining a final analytical method (Figure 1C), 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.