2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.

This article was originally published in Electrophoresis 2011, 32, 1292–1301, DOI

4360 2-Dimensional difference gel electrophoresis (2-D DIGE) is a method that circumvents the gel-to-gel variability inherent in conventional 2-dimensional gel electrophoresis (2-DE). We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The FVIII heterodimer is composed of heterogenous, strongly glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments and their identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII’s glycans due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate BAX 855, a PEGylated longer-acting variant of recombinant FVIII. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool to characterize and control quality of very complex pharmaceutically active ingredients such as PEGylated proteins. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment. Monetti:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Weber:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.