LC/MS Charge Variant Characterization of an ECD-Fc Fusion Protein using OFFGEL and SAX Fractionation Workflows

摘要

Receptor extracellular domain fusion proteins represent a growing class of biotherapeutics. Since these fusion proteins can be highly glycosylated they have broad distributions of charge variants. In todays biotechnology industry there is an increasing need to structurally characterize the charge heterogeneity of these variants by methods other than electrophoresis. In this study we have developed rigorous analytical workflows to structurally characterize an ECD-Fc fusion protein using both pI based separation (OFFGEL fractionation) and IEX chromatography to fractionate the protein heterogeneity upstream. We have found that the glycosylation in the ECD region is the major contributor to the overall charge heterogeneity. These glycosylated species have been fractionated and structurally characterized using OFFGEL fractionation and Strong Anion Exchange (SAX) chromatography workflows followed by LC/MS.