This technical note demonstrates the powerful capabilities of an imaged capillary isoelectric focusing (icIEF)-UV/MS workflow for charge heterogeneity analysis of human transferrin (Tf), an acidic glycoprotein detected in an isoelectric point (pI) range of 5.5-6.5. The icIEF-UV/MS workflow provides high-resolution charge separation, sensitive detection and confident identification of the Tf glycoforms and charge variants. The coupling of online icIEF separation with accurate mass measurement eliminates the need for labor- and time-intensive fraction collection followed by offline MS analysis, significantly reducing the time to obtain the results from weeks to hours for routine samples.
Charge heterogeneity analysis of protein therapeutics is important for product quality attributes (PQAs) assessment to ensure the quality, safety and efficacy of drug molecules from the development to quality control. Traditional icIEF workflows can provide reproducible charge variant separation for PQA analysis in different biopharmaceutical settings.1 However, it is challenging for these approaches to identify the proteoforms under each charge-variant peak or elucidate complex charge profiles without the mass information. The icIEF-UV/MS workflow offered by the Intabio ZT system overcomes these challenges by coupling high-resolution icIEF separation with accurate mass measurement. This powerful workflow has proven to be highly effective for charge heterogeneity analysis of protein therapeutics with a wide pI range of 7.3-9.1.
In this technical note, the capability of icIEF-UV/MS was extended to sialic acid-containing Tf. The streamlined icIEF-UV/MS workflow provides reproducible charge variant separation and confident identification of the Tf glycoforms containing different numbers of sialic acids and charge variants carrying various post-translational modifications (PTMs).
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