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April 01, 2014 (Vol. 34, No. 7)

Mastering Charge Heterogeneity Analysis of Therapeutic Proteins

  • 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

    Click Image To Enlarge +
    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.

    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.

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