April 1, 2014 (Vol. 34, No. 7)

Analyzing charge variants of biopharmaceuticals is a critical component of product development and quality control. Charge variants commonly occur as a result of both chemical and post-translational modifications including deamidation, oxidation, glycosylation, and glycation. These changes can affect biological activity, patient safety, and drug stability.

Charge variants have traditionally been monitored by ion exchange chromatography (IEC), but newer CE-based techniques, such as imaged capillary isoelectric focusing (icIEF), offer the advantages of generic methods for multiple products and faster analysis times. Analysis of biopharmaceuticals using icIEF on the iCE3 system provides high-resolution charge heterogeneity peak profiles in 10 minutes. In this tutorial, we describe a simple and easy approach for icIEF method development on the iCE3 along with tips to improve method robustness.

Imaged cIEF Principle

Significantly different from traditional cIEF, the iCE3 performs capillary IEF with whole-column detection, which eliminates the need for a lengthy mobilization step—this both increases sample throughput and reduces assay complexity. Prepared samples for icIEF contain a mixture of the protein of interest, carrier ampholytes, and pI markers.

When this sample mixture is injected, it fills the entire capillary cartridge, where separation takes place. Two electrolytic tanks at each end of the cartridge are filled with acid (anolyte) and base (catholyte). Samples are focused by applying voltage across the cartridge, and during the focusing step, a pH gradient forms across the capillary

The pI markers and protein of interest migrate through the capillary until they reach a pH value where their net charge is zero—this is their isoelectric point. The iCE3 then uses whole-column imaging detection at 280 nm to capture the separation within the capillary. Finally, the capillary is washed to ready it for the next sample injection. The full process from sample injection through final wash takes place in 10–12 minutes.

Method Development

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.

Figure 1. 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.

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 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 2A 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 2B 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.

Figure 2. (left) The separation of IgG 1 Kappa demonstrates the adverse effects of salts (A). Triplicate runs at 100mM NaCl show that salt also affects reproducibility (B).

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 (

As with all separation techniques, high-quality reagents should be used with icIEF methods to ensure consistent results (Figure 3B). Using improperly stored or expired consumables and reagents can also have a profound effect on performance. This is especially true for methods that employ urea to eliminate aggregation. Urea solutions should be made fresh and kept away from heat to avoid thermal degradation. One of the thermal degradation products of urea, isocyanic acid, will rapidly react with amine groups and artificially increase a protein’s acidic species’ percent composition (Figure 3A).

Figure 3. (right) High-quality reagents help ensure consistent results. (B) For example, if urea solutions are used, they should be made fresh and kept away from heat. Doing so can avoid artificial increases in a protein’s acidic species’ percent composition (A).


The iCE3 system’s quick and easy icIEF method development lets even those analysts new to icIEF develop robust charge heterogeneity methods in an afternoon by following some simple procedures and guidelines. Potential issues that can arise with commonly interfering sample matrix components are easily resolved through addition of solubilizers, dilution, or buffer exchange. In addition, the high-resolution, 10-minute separations obtained are ideal for the characterization and monitoring of charge variants in biopharmaceutical formulations.

Scott Mack ([email protected]) is a senior R&D scientist and Susan Darling is a director of marketing at ProteinSimple.

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