PEGylation of therapeutic proteins has significantly improved their value by altering the physicochemical and biological properties such as increased solubility, decreased immunogenicity, increased half-life, and protection against proteases. Size Exclusion Chromatography (SEC) is the method of choice for determining impurities with molecular mass higher than that of PEGylated proteins. SEC of PEGylated proteins poses a significant challenge due to PEG-mediated interaction with silica stationary phases, leading to lower recovery, poor peak shape, and undue tailing. This application note describes a simple and sensitive SEC method for determining the purity of PEG Granulocyte Colony Stimulating Factor (PEG GCSF). Separation and quantification of PEG GCSF was achieved using an Agilent AdvanceBio SEC, 130Å, 7.8 × 300 mm, 2.7 μm column employing aqueous mobile phase. An excellent correlation coefficient was observed for the linearity curve in the range 12.5 to 2,000 µg/mL, indicating that the method is quantitative. Retention time and peak area precision of the method were excellent, demonstrating the suitability of the method. Furthermore, AdvanceBio SEC was able to separate and quantify aggregates obtained by forced-stress studies.
Publication number: 5991-6791EN
Publication Date: March 23, 2016