Capillary isoelectric focusing has in recent years increasingly been applied as one of the main analytical methods for determination of protein charge and isoelectric point. It is one of the key tools for monitoring charge heterogeneity, especially in biopharmaceutical industry. However, analysis of fusion proteins can often be challenging due to their structural complexity. As a result, acquired electropherograms may exhibit irreproducible and/or unspecific peaks, which are caused by protein aggregation and precipitation. Therefore, it is crucial to achieve sufficient solubility in the capillary. The aim of the master's thesis was to expose the drug substance to different solubilizers in various concentration (urea, formamide, sucrose, zwitterion detergent CHAPS, and zwitterion NDSB in combination with taurine). In some cases where separation has been partially successful (e.g., formamide and sucrose), results have been improved by decreasing protein concentration. To make separation easier, we enzymatically removed negatively charged sialic acids, which are linked to the protein molecule. Acquired electropherograms are consequently shifted to basic side of pH gradient and have fewer peaks. The most successful separation was achieved by analyzing 0,4 mg/mL of fusion protein in 3 M urea and focusing time of 12 minutes; every other concentration of urea and other tested solubilizers did not produce satisfactory resolution or reproducibility.
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